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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from authoritative database
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Chemical name of vehicle: Dimethylformamide, Hydrogenated castor oil (HCO-40)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 microL/L [ mixed solution of Dimethylformamide / Hydrogenated castor oil (HCO-40) = 3/1 (w/w) ]


Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Test temperature:
20 ± 1° C
Nominal and measured concentrations:
Test chemical concentrations used for the study were 0 (control), 0 (vehicle control), 0.022, 0.046, 0.10, 0.22, 0.46 mg/L (geometric ratio: 2.2)
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: Test solution volume 80 mL per vessel
- Renewal rate of test solution (frequency/flow rate): Total amount of the test solution was renewed every 24 hours
- No. of organisms per vessel: 10 per concentration (1 per vessel)
- No. of vessels per concentration (replicates): 10 replicates
- No. of vessels per control (replicates): 10 replicates
- No. of vessels per vehicle control (replicates): 10 replicates

OTHER TEST CONDITIONS:
- Photoperiod: 16 hours light / 8 hours dark


Reference substance (positive control):
not specified
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 0.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 50 % Lethal concentration (LC50) to parental Daphnia

- 50 % Lethal concentration (LC50) to parental Daphnia (21 d): > 0.46 mg/L

- Lowest observed effect concentration (LOEC) for reproduction (21 d): 0.46 mg/L

Validity criteria fulfilled:
not specified
Conclusions:
After the exposure of test chemical with the aquatic invertebrates daphnia magna for 21 days, median effect concentration EC50 was determined to be 0.46 mg/l and the 21 d NOEC was 0.22 mg/l.
Executive summary:

Long term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical. The study was performed in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. Test chemical solution was prepared in vehicle Dimethylformamide, Hydrogenated castor oil (HCO-40)  (100 microL/L). Test chemical concentrations were verified analytically using GC/MS. Test chemical concentrations used for the study were 0 (control), 0 (vehicle control), 0.022, 0.046, 0.10, 0.22, 0.46 mg/L (geometric ratio: 2.2), respectively. 10 daphnids/conc. (1 per vessel) were exposed to test chemical in test vessel for 21 days. Volume of test solution in test vessel was 80 ml. Total amount of the test solution was renewed every 24 hours. Controls were also run simultaneously during the study. Test conditions involve a temperature of 20 ± 1° C and under a photoperiod of 16 hours light / 8 hours dark.  All experiments were performed in 10 replicates. After an exposure period of 21 days, the reproduction effect of the test organism was noted. On the basis of the effect of test chemical on reproduction rate of the test daphnids, the 21 d NOEC, EC50 and LC50 value was determined to be 0.22, 0.46 and >0.46 mg/l, respectively. Based on the result, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

Description of key information

Long term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical. The study was performed in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. Test chemical solution was prepared in vehicle Dimethylformamide, Hydrogenated castor oil (HCO-40)  (100 microL/L). Test chemical concentrations were verified analytically using GC/MS. Test chemical concentrations used for the study were 0 (control), 0 (vehicle control), 0.022, 0.046, 0.10, 0.22, 0.46 mg/L (geometric ratio: 2.2), respectively. 10 daphnids/conc. (1 per vessel) were exposed to test chemical in test vessel for 21 days. Volume of test solution in test vessel was 80 ml. Total amount of the test solution was renewed every 24 hours. Controls were also run simultaneously during the study. Test conditions involve a temperature of 20 ± 1° C and under a photoperiod of 16 hours light / 8 hours dark.  All experiments were performed in 10 replicates. After an exposure period of 21 days, the reproduction effect of the test organism was noted. On the basis of the effect of test chemical on reproduction rate of the test daphnids, the 21 d NOEC, EC50 and LC50 value was determined to be 0.22, 0.46 and >0.46 mg/l, respectively. Based on the result, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.22 mg/L

Additional information

Experimental studies of the test chemical and various supporting weight of evidence studies for its structurally similar read across chemical were reviewed for long term toxicity to aquatic invertebrate end point which are summarized as below:

 

In an experimental study from authoritative database, long term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical. The study was performed in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. Test chemical solution was prepared in vehicle Dimethylformamide, Hydrogenated castor oil (HCO-40)  (100 microL/L). Test chemical concentrations were verified analytically using GC/MS. Test chemical concentrations used for the study were 0 (control), 0 (vehicle control), 0.022, 0.046, 0.10, 0.22, 0.46 mg/L (geometric ratio: 2.2), respectively. 10 daphnids/conc. (1 per vessel) were exposed to test chemical in test vessel for 21 days. Volume of test solution in test vessel was 80 ml. Total amount of the test solution was renewed every 24 hours. Controls were also run simultaneously during the study. Test conditions involve a temperature of 20 ± 1° C and under a photoperiod of 16 hours light / 8 hours dark.  All experiments were performed in 10 replicates. After an exposure period of 21 days, the reproduction effect of the test organism was noted. On the basis of the effect of test chemical on reproduction rate of the test daphnids, the 21 d NOEC, EC50 and LC50 value was determined to be 0.22, 0.46 and >0.46 mg/l, respectively. Based on the result, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

In a supporting weight of evidence study, chronic toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical (secondary source, 2006). The study was performed following the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Modification with the OECD TG is that aeration of exposure media was done. Test solutions were prepared daily by stirring the test substance in test media under slow stir conditions (21 h) in sterilized mixing vessels. The mixing vessels were cylindrical brown glass bottles with teflon covered screw caps, fitted with a drain port near the bottom for drawing off the test solution. The volume of the mixing vessels was 2 L. After stirring, the contents of the vessels were left to settle for 2 h. The saturated aqueous phase was then taken out of the drain port. The first fraction 0-100 mL was discarded. The fraction between 100 and 1800 mL was used for rinsing (200 mL) and filling (1000 mL) the test flasks for toxicity testing and for analytical measurements (500 mL), if done. Rinsing of the test vessels was carried out to saturate the surfaces of the test vessels. After filling, the vessels were closed immediately by using autoclaved silicone stoppers and only opened to introduce the test organisms and again at the renewals of the test media. The test media were not stored for more than 1 - 2 hours prior to testing. Daphnia magna STRAUS (Water flea) of 4 to 24 hr old was used as a test organism. Test organisms was bred in the laboratory of the Fh-IME (testing facility). The Daphnia magna were fed at each renewal with suspensions of unicellular green algae. The suspensions of Desmodesmus subspicatus (daily prepared from axenic cultures) were controlled analyzed for microbial contamination one and two weeks after test start by using "Cult-Dip combi® Dip Slides (Merck)". No bacterial contamination was detected. The content of food in the test suspensions, measured as turbidity at 758 nm, increased during the test from 7 mg C/L equivalents to 15 mg C/L equivalents. Test chemical concentrations used for the study were 0, 0.025, 0.069, 0.185 and 0.500 mg/l (nominal conc.); < Limit of quantification, 0.02, 0.056, 0.163 and 0.534 mg/l (measured initial conc.) and < Limit of quantification, 0.003, 0.005, 0.014 and 0.095 mg/l (geometric measured conc.), respectively. All the test concentrations were sampled for chemical analysis three times a week at renewal of the test media. A 500 mL aliquot of the fresh solutions was used for analysis. After 24 h, at the next renewal, the aged test liquids were pooled (vessels 1- 5 and 6-10) and analysed. The analyte was extracted from the aqueous test samples by liquid-liquid partitioning with n-hexane. After derivatization of the analyte by MSTFA measurement was performed by GC-MS using n-dodecanol-d25 as internal standard. The method was validated for the determination of the test item in Daphnia test medium in the concentration range of 1.0 - 100 μg/L. Test daphnids (10 organisms) were exposed to test chemical in test vessel for an exposure period of 24 hrs. ll test vessels were aerated with sterile filtrated synthetic air: the autoclaved silicone stoppers were fitted with fine glass capillaries connected to the aeration unit. The aeration was necessary to avoid severe oxygen depletion due to the increase of transferred bacteria with growing Daphnia magna as observed in pre-studies and the associated oxygen consumption by the degradation of the test substance. Test conditions involve a photoperiod of 16/8 hour light: dark condition, temperature of 21 to 22°C, photoperiod of 9.3 to 9.5, the oxygen saturation never fell below 56 % (4.0 mg/L) and light intensity of 585 to 647 lux, respectively. The parent Daphnia magna were assessed visually daily for immobility and any other abnormalities in appearance and behaviour. At study termination, the length of the adults was measured by digital photography and image analysis and their statistics compared with those of the control animals. The newborn Daphnia magna in each beaker were counted at each daily renewal of the test solutions, inspected for abnormalities in condition, and removed. The evaluation of the concentration-effect relationships and the calculations of effect concentrations were based on mean measured initial concentrations as multiple peak concentrations, as well as on geometric means between mean measured initial and aged (24h) test concentrations. For each endpoint, the NOEC, LOEC, and, if possible, the EC50, EC20 and EC10 were determined. A LOEC and NOEC were calculated by ANOVA followed by Williams’ test or an appropriate nonparametric test suggested by the ToxRat program. When the test results showed a concentration-response relationship, the data were analysed by regression using Probit-analysis assuming log-normal distribution of the values using the computer program ToxRat program. Based on the effect on reproduction rate of the test daphnids, the 21 d NOEC, LOEC, EC10 and EC20 value was determined to be 0.014, 0.095, 0.013 and 0.034 mg/l (measured geometric conc.) and 21 d NOEC & LOEC value was determined to be 0.185 and 0.5 mg/l (nominal concentration), respectively. Based on the result (21 d NOEC = 0.185 mg/l), test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

For the test chemical, long term toxicity to aquatic invertebrate study was carried out for 21 days. Study was performed following the principles of the OECD test guideline 202, part 2 (from handbook, 2009 and secondary source, 1995). Test chemical concentrations were measured by DOC analysis. Total 20 daphnids (5 per test vessel) were exposed to different test chemical conc. (i.e., 1, 3, 10, 30, and 100 mg/l) for a period of 21 days under flow through conditions. Test solutions were changed three times per week. The test vessels are controlled regularly and the time of first appearance of decedents and the number of juvenile organisms was determined. Measured (DOC) concentrations were 0.6/0.5 mg/l at 0 hours, 1.3/1.2 mg/l at 48 hours, and 1.9/1.8 mg/l at 120 hours for the 10 mg/l group. Similar measured concentrations were obtained in the 30 mg/l and 100 mg/l groups. No information was given on possible physical effects on the Daphnia (surface floating, etc.). At (nominal) concentrations of 1 mg/l no effects were noted, while at concentrations of 3 mg/l and above, fertility was significantly effected. There were no effects on the adults in any group including controls on day 2. On day 5, 1/(20) died in the control group, and 8/(20) in the 10 mg/l group, 4/(20) in the 30 mg/l group, and 1/(20) in the 100 mg/l group. By day 21, adult mortality reached or exceed 50% in all test groups above 3 mg/l, while being 10% in the control and low dose groups. Thus, on the basis of these effects, the 21 d NOEC and LOEC value was determined to be 1.0 mg/l and 3.0 mg/l, respectively.  Thus, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Another chronic toxicity to aquatic invertebrate study (Kuhn R et. al., 1989) was carried out for assessing the effect of test chemical. The study was performed in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test) and Federal Environment Agency (1984), respectively. The test substance was dispersed to make up a stock solution of 200 mg/L, then gradual serial dilutions (corresponding to a ratio of 1:2) of the stock solution were made to produce the concentration range tested. Samples were taken twice from selected concentration levels of the test series during the test period and analysed chemically. The first sampling was taken before the 7th day previous to any offspring appearance, the second sample was taken between day 16 and day 21. No further details on analytical methods were presented. Test organism Daphnia magna (Water flea) was maintained in accordance with the procedure practised since 1978. During the acclimation period, test organism was fed daily with dry algae Scenedsmus spp. 9g of feed were suspended in 1000 mL tap water and 2 mL were added to each 2 L cultures. Test daphnids (5 test organisms/vessel) were exposed to test chemical conc. (0.4 to 50 mg/l) in 400 ml glass beakers filled with 250 mL test water.  Deionised water was used to prepare standard artificial medium (synthetic fresh water) as stated by DIN - German Institute of Standardisation. Aeration was not provided in the test vessel. All experiments were performed in 4 repilcates. Test conditions involve a temperature of 25 +/- 1°C, pH of > 7, dissolved oxygen >69%, light with fluorescent lamps - Philis TL having intensity of 40/25W. Mortality, reproduction rate and appearance of offspring, daily. Student's t-test and U-test was used for deterimining the NOEC of reproduction rate and parent mortality. On the basis of the effect on reproduction rate of the test organism, the 21 day NOEC value was determined to be 1.0 mg/l.

 

On the basis of the above results, it can be concluded that the test chemical test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.