Chemex Environmental International Ltd

Freshwater Testing Services

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An extensive range of acute and chronic freshwater toxicity studies can be conducted at Chemex. An extensive range of acute and chronic freshwater toxicity studies can be conducted at Chemex. Species available for testing include algae (Pseudokirchneriella subcapitata, Scenedesmus subspicatus and Anabaena flos-aquae), invertebrate (Daphnia magna and Chironomus riparius), fish (Rainbow Trout and Carp) and the aquatic plant Lemna minor. In support of the above studies, Chemex also offer a comprehensive Analytical Support service utilising the on-site analytical facility equipped with a range of analytical techniques.

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Algal Growth Inhibition (OECD 201)

Freshwater Algal Growth Inhibition Test (OECD 201, JMAFF, EC C3)
The Chemex preferred species for use in this freshwater algal growth inhibition study is Pseudokirchneriella subcapitata, although other species can be offered on request. This test guidelines for this study are OECD 201, Japanese MAFF & EC C3.

The growth inhibition test is performed semi-axenically (i.e. minimising contamination by other organisms) in glass flasks under controlled conditions.  Growth of the alga is determined by cell counting or spectrophotometry at 24-hour intervals for periods of exposure up to 96 hours. The data are used to determine the LC50 value for growth-rate. The highest no effect concentration (NOEC) is determined using appropriate statistical procedures

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Anaerobic Biodegradation (ISO/DIS 11734 / OECD 311)

Freshwater Anaerobic Biodegradation Study
This method is intended to provide a simple and reliable screening test for the ultimate biodegradability of chemicals under anaerobic conditions. The method is applicable to soluble and poorly soluble organic chemicals.

A known volume of anaerobic sludge (corresponding to 10% of the sludge concentration in a real digester) suspended in an oxygen free medium is placed in a suitable vessel with a headspace into which any gases produced may be evolved. Prior to sealing a small amount of test compound is added. The vessels are incubated at constant temperature (35 ± 1°C) and pH for a period of 8 weeks. The headspace pressure, resulting from the production of gas, is measured.

From the measured values of net gas production the extent of biodegradation is calculated. The kinetics of the degradation are followed by intermediate measurements at suitable intervals during the course of the test.

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BODIS in Seawater (PARCOM)

Biodegradability of Insoluble Substances (BODIS) in Seawater (PARCOM)
This method was developed for the assessment of degradation of poorly soluble materials in the marine environment, however, it can also be adpated for Freshwater purposes. The test vessels are closed glass bottles with a known volume of natural seawater (2/3) and air (1/3). They are shaken continuously to ensure steady state oxygen partitioning between liquid and gas phases. The degradation is followed by weekly measurements of the biochemical oxygen demand (BOD) in the aqueous phase for a 28 day period.

The BOD is calculated from the cumulative oxygen uptake.

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Daphnia Acute Immobilisation (OECD 202)

Daphnia magna Acute Immobilisation Test - Static or Semi-static (OECD 202, JMAFF, EC C2, DTAPS)
Juveniles of the cladoceran crustacean Daphnia magna of less than 24 hours old are exposed to a range of concentrations of test substance or effluent in water. Daphnia are observed after 24 and 48 hours and the number immobilised in each vessel is recorded.

Results are expressed as EC50 values (concentrations causing 50% of the Daphnia to be immobilised) for 24 and 48 hours exposure.  Classification is based on the 48 hour EC50 figure. The maximum no effect concentration (NOEC) and minimum concentration resulting in significant (P >= 0.05) immobility (LOEC) are also reported.

This study can be performed under static or semi-static conditions and follows guidelines OECD 202, Japanese MAFF, EC C2 & DTAPS.

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Daphnia magna Reproduction (OECD 211)

Daphnia magna Reproduction Test - 21 Day - Semi-static
The primary objective of the test is to assess the effect of chemicals on the rate of reproduction of Daphnia magna. The concentrations used in the study are based on the results of an acute immobilisation test. The duration of the study is 21 days and the number of offspring produced, together with adult and juvenile survival, is reported. A semi-static system is recommended and the frequency of test media renewal depends on the stability of the substance. The study is started with juveniles which are female and start to produce live young by parthenogenesis after about 7 days.

Juvenile production is compared to that of the controls to determine the lowest observed effect concentration (LOEC) and the no effect concentration (NOEC) in addition of the EC50. Other adverse effects are recorded such as abnormal development of juveniles in the brood pouch (white eggs). Shedding of un-hatched eggs, presence of male Daphnia, ephippial eggs and differences in the size of adults at the end of the test are also recorded.

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DOC Die-Away Test (OECD 301A / EC C.4.3)

Freshwater Biodegradation - DOC Die-Away Test
The loss of dissolved organic carbon (DOC) is followed by analysis every few days during the 28 day study period and is compared with the total organic carbon for the known weight of test substance. A sodium acetate reference is used and greater than 60% loss is expected.

This method is similar to the Modified OECD Screening Test (OECD 301E) but uses a higher concentration of micro-organisms.

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Effects on Sediment Dwelling Organisms - Chironomid (OECD 218)

Chironomid Toxicity Using Spiked Sediment - Freshwater
This Test Guideline is designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae of the freshwater dipteran Chironomus sp.

First instar chironomid larvae are exposed to at least five concentrations of the test chemical in sediment - water systems. The test substance is spiked into the sediment and first instar larvae are subsequently introduced into test beakers in which the sediment and water concentrations have been stabilised. Chironomid emergence and development rate is measured at the end of the test. The maximum exposure duration is 28 days for C. riparius, C. yoshimatsui, and 65 days for C. tentans. The limit test corresponds to one dose level of 1000 mg/kg. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). The study report should include the development time and the total number of fully emerged midges (sex and number are recorded daily), the observation of any abnormal behaviour the number of visible pupae that have failed to emerge and any egg masses deposition. The data are analysed either by using a regression model in order to estimate the concentration that would cause x % reduction in emergence or larval survival or growth, or by using statistical hypothesis testing to determine a NOEC/LOEC.

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Effects on Sediment Dwelling Organisms - Chironomid (OECD219)

Chironomid Toxicity Using Spiked Water - Freshwate
This study is designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae of the freshwater dipteran Chironomus sp.

First instar chironomid larvae are exposed to at least five concentrations of the test chemical in sediment-water systems. The test starts by placing first instar larvae into the test beakers containing the sediment-water system and subsequently spiking the test substance into the water. Chironomid emergence and development rate is measured at the end of the test. The maximum exposure duration is 28 days for C. riparius, C. yoshimatsui, and 65 days for C. tentans. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). The study report should include the development time and the total number of fully emerged midges (sex and number are recorded daily), the observation of any abnormal behaviour, the number of visible pupae that have failed to emerge and any egg masses deposition. The data are analysed either by using a regression model in order to estimate the concentration that would cause x % reduction in emergence, larvae survival or growth, or by using statistical hypothesis testing to determine a NOEC/LOEC.

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Fish Acute Toxicity (OECD 203)

Freshwater Fish, Acute Toxicity Test (OECD 203, JMAFF, EC C1)
The preferred freshwater species are rainbow trout (Oncorhynchus mykiss) or carp (Cyprinus carpio).

Fish are introduced into glass aquaria containing water to which the test substance has been added at known concentrations. Fish are observed for mortality and sub-lethal symptoms at specified periods of time. The results of the test are expressed in the form of LC50 values (that is the concentration causing 50% mortality among the test population).

The duration of exposure is normally 96 hours. The maximum concentration at which no mortality occurs (NOEC) and the minimum concentration at which 100% mortality occurs are also reported together with observations of toxic symptoms. The slope of the dose/response curve is provided wherever possible. This provides useful extra information for use in environmental risk assessment or hazard classification of the test substance.

In order to reduce the numbers of fish tested, but still informing the hazard-assessment process, the fish test is now commonly conducted as a limit test with a single concentration tested. The lowest concentration causing effects in studies previously conducted with algae and invertebrates is used to demonstrate the LC50 for fish is below that concentration, therefore demonstrating that the fish is not the most sensitive of the organisms tested. Where mortalities are seen a full range of concentrations is tested to determine the LC50.

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Fish Early Life Stage Toxicity (OECD 210)

Freshwater Fish - Early Life Stage Toxicity (FELST) - OECD 210
The preferred freshwater species are rainbow trout (Oncorhynchus mykiss) or carp (Cyprinus carpio).

The early life stages of fish are exposed to the chemical usually under flow-through conditions. The study starts by placing fertilised eggs in a series of replicate test chambers and continues until all the fish are free-swimming and feeding exogenously. Lethal and sub-lethal effects such as growth and development are assessed and compared with un-exposed control replicates. The larval and early fry stages are usually the most sensitive stages to the effects of xenobiotics.

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Freshwater Fish Bioconcentration (OECD 305)

Freshwater Bioconcentration: Flow-through Fish Test
The preferred freshwater species are rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio).

The objective of this study is to determine the steady-state bioconcentration factor (BCFss). Fish are exposed under flow-through conditions to water of known quality to which the test substance has been added at known concentrations for a period normally not exceeding 28 days (uptake phase). After the uptake phase the remaining fish are transferred to clean tanks under flow-through conditions for a further period of time normally not exceeding 14 days (depuaration phase). Samples of fish and water are analysed for the test material at predetermined time points in both test phases.

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Freshwater Fish Juvenile Growth Test (OECD 215)

28 Day - Juvenile Fish Growth Test (Oncorhynchus mykiss)
This study is designed to assess the effects of prolonged exposure to chemicals on the growth of juvenile fish.

Juvenile fish in exponential growth phase are weighed, then placed in test chambers and are normally exposed to five sublethal concentrations of the test substance dissolved in water preferably under flowthrough, or, if not possible, under appropriate semi static (static-renewal) conditions. The test duration is 28 days. The daily food ration is based on initial fish weights and may be recalculated after 14 days.

Measurement of the concentration of the test substance should be included in the report, along with the observation of external abnormalities and abnormal behavior and the fish weighting at the end of the test. Effects on growth rates are analyzed using a regression model in order to estimate the concentration that would cause a x % variation in growth rate, i.e. ECx. Alternatively, the data may be compared with control values in order to determine the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC).

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Lemna Growth Inhibition (OECD 221)

Freshwater - Lemna minor Growth Inhibition Test
Cultures of the floating aquatic plant Lemna minor are exposed to a range of concentrations of test substance or effluent under defined conditions light and temperature. The inhibition of growth, relative to unexposed controls, is determined in a static, or semi-static system over 7 days. The data are used to determine the 7 day LC50 (the concentration causing a 50% inhibition of growth rate as measured by the rate of increase in frond numbers) and the NOEC for growth rate. Effects such as chlorosis, abnormal colony formation and root formation are also recorded.

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Manometric Respirometry (OECD 301F)

Freshwater Manometric Respirometry Test (Oxitop) - Ready Biodegradability
This freshwater ready biodegradibility test uses Oxitop apparatus and is part of the OECD 301 series of methods.

A measured volume of inoculated mineral medium, containing a known concentration of the test substance (100 mg/l giving at least 50-100mg ThOD/l) as the source of organic carbon, is stirred in a closed flask at a constant temperature for up to 28 days. During the course of the test oxygen is consumed and carbon dioxide evolved. The carbon dioxide is absorbed in sodium hydroxide and the drop in pressure in the test vessels therefore determines the oxygen consumption. The caps of the test vessels contain a pressure transducer and microprocessor to measure and calculate BOD.

The amount of oxygen taken up by the microbial population during biodegradation of the test substance is expressed as a percentage of ThOD or, less satisfactorily, COD.

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Modified Sturm Test (OECD 301B)

Freshwater Modified Sturm Test (OECD 301B / EC C.4.3)
This method is suitable for non-volatile substances that may be poorly soluble in water.

The amount of carbon dioxide (CO2) produced as a percentage of theoretical yield (based on total organic carbon analysis) is used as a basis for assessing whether the material biodegrades. CO2 is measured by way of a sodium hydroxide trap. The study is run for a minimum of 28 days and may be continued if the yield of CO2 is showing signs of increase towards the end of the 28 day period.

A positive control of sodium acetate is run alongside and a degradation of greater than 60% over 28 days should be seen. A blank control containing just the inoculum and nutrient mixture is used to check background levels of activity.

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OECD Biodegradation Screening (OECD 301E)

Freshwater Modified OECD Screening test (OECD 301E / EC C.4.2)
The loss of dissolved organic carbon (DOC) is followed by analysis every few days during the 28 day study period and is compared with the total organic carbon for the known weight of test substance. A sodium acetate reference is used and greater than 60% loss is expected.

This method is similar to the DOC Die-Away Test (OECD 301A) but employs a relatively low concentration of micro-organisms.

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Ready Biodegradability (ISO 14593 / OECD 310)

Freshwater Ready Biodegradability – CO2 Headspace Test
This method is based on ISO 14593 / OECD 310 : ‘Carbon dioxide (CO2) Headspace Biodegradation Test’ and provides a test for assessing the ultimate, aerobic biodegradability of organic substances in the freshwater environment. It provides similar information to the methods described in OECD 301.

The test is performed in sealed bottles with a headspace of air that provides a reservoir of oxygen (O2) for aerobic biodegradation. CO2 evolution from the ultimate aerobic biodegradation of the test substance is determined by measuring the inorganic carbon (IC) produced in the test bottles over that produced in blanks that contain inoculated medium only. The ultimate aerobic biodegradation is the breakdown of an organic chemical by micro-organisms in the presence of O2 resulting in the production of CO2, water and mineral salts (ie mineralisation) and microbial cellular constituents (biomass).

The extent of biodegradation is then expressed as a percentage of the theoretical maximum IC production (ThIC), based on the quantity of test substance (as total organic carbon) added initially. The test is typically run for 28 days.  Values =60% ThIC in a 10 day window demonstrates ready biodegradability.

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Zahn-Wellens/EMPA Test (OECD 302B)

Freshwater Modified Zahn-Wellens/EMPA Test
This freshwater method is not suitable for volatile materials.

The test material is added to a large volume of activated sludge and nutrient mixture, and the decline in dissolved oxygen concentration measured frequently. The duration of the study is generally 28 days but may be extended if a significant lag period is noted at the start. A result of > 70% degradation means that the substance is ‘ultimately’ biodegradable but < 70% and > 20%, it is ‘inherently’ biodegradable.

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Chemex can offer a range of studies relevant to your REACH Submissions
Chemex can offer a number of studies that will be relevant to your REACH submissions.  You can rest assured that we have the systems, staff and facilities in place to conduct studies for REACH purposes to a very high standard.

REACH is a significant driving force in the work that Chemex conducts, including Physico-Chemical, Biodegradation, Bioaccumulation, Aquatic Toxicology and Terrestrial Toxicology, all to GLP Compliance.

Chemex remain at the forefront of Ecotoxicology testing, supplying high quality data at competitive prices.

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