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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Concentrations: 1.79 mg/L, 3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L

- Sample storage conditions before analysis: sample was analysed immediately after sampling from each test concentration
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test chemical of 100 mg was dissolved in 100 ml of test solution to getstock solution of 1000 mg/L, which was analytically determined to identify the real concentrations.
- Controls: M7 Medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: straus
- Age at study initiation (mean and range, SD): <24 hours and neonates obatined form second brood pouch of parent daphnia
- Stage and instar at study initiation: Neonates
- Age of parental stock (mean and range, SD): > 3weeks
- Feeding during test : no feed was provided during feed
- Food type: parents were feed with green agae during culture maintenance
- Frequency: daily

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
140 mg/L CaCO3
Test temperature:
21.7±0.1 °C
pH:
7.2 to 7.5
Dissolved oxygen:
7.0 to 7.9
Nominal and measured concentrations:
Concentrations: 1.79 mg/L, 3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 25 ml Glass beaker, 5 ml, fill volume 20 ml
- Volume of solution: 20 ml
- Aeration: no aeration was provided
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5 organism per vessel
- No. of vessels per concentration (replicates):4 replicate per concentration
- No. of vessels per control (replicates): 4 replicates per control
- No. of vessels per vehicle control (replicates): not applicable



OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted
- Photoperiod: 16 hour light and 8 hour dark
- Light intensity: illumination: 1000 – 1500 Lux,

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: range finding study was not coducted, definitive test was performed based on the available target data
Reference substance (positive control):
yes
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
6.62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% 4.22-11.86
Details on results:
- Behavioural abnormalities: None
- Other biological observations:
- Mortality of control: No moratality in control was observed
- Other adverse effects control:
- Immobilisation of control: no immobilisation
Results with reference substance (positive control):
- Results with reference substance valid? Yes valid and EC 50: 0.831
Reported statistics and error estimates:
The EC50 was caluculated using probit method, by SYS stat pro

Immobilisation of daphnia:

Test concentration mg/L

Immobilisation 0 hr

Immobilisation 24hr

Immobilisation 48 hr

Total Percent immobilisation

Control

0/20

0/20

0/20

0

1.79

0/20

0/20

1/20

5

3.04

0/20

3/20

1/20

20

5.17

0/20

3/20

4/20

35

8.79

0/20

6/20

4/20

50

14.9

0/20

10/20

19/20

95

 

Test Concentration(mg/L)

 

pH

 

Dissolved oxygen

 

Temperature °C

 

0 Hour

 

24

Hours

48 Hours

 

0 Hour

 

24

Hours

48 Hours

 

0 Hour

 

24

Hours

48 Hours

 

Control

7.2

7.5

7.2

8.7

7.1

6.9

21.4

221.6

21.8

 

1.79

7.4

7.5

7.4

8.5

7.3

6.8

21.3

21.6

21.9

 

3.04

7.5

7.6

7.5

87.5

7.7

6.8

21.3

21.7

21.9

 

5.19

7.4

7.4

7.2

8.4

7.1

6.4

21.3

21.8

21.9

 

8.79

7.3

7.4

7.2

8.4

7.1

6.0

21.2

21.5

21.9

 

14.9

7.3

7.5

7.5

8.3

7.2

5.9

21.2

21.5

21.9

Validity criteria fulfilled:
yes
Conclusions:
The 48 hours EC50 concentration of test chemical was reported to be 6.32 mg/L (4.22-11386).
Executive summary:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The saturated test solution was prepared by dissolving 100mg of test chemical in 100ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 990.12 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 1.79 mg/L,  3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L, respectively was from the saturated test concentration. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 6.32 mg/L (4.22-11386). Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.

Description of key information

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 990.12 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 1.79 mg/L,  3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L, respectively was from the saturated test concentration. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 6.32 mg/L (4.22-11386). Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
6.22 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 short term toxicity to aquatic invertebrate end point which are summarized as below:

 

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 990.12 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 1.79 mg/L,  3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L, respectively was from the saturated test concentration. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 6.32 mg/L (4.22-11386). Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.

In an experimental study from study report, an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp., Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 100.0 mg/l was prepared by dissolving dark orange powder in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Study was performed using 5 organisms per vessel/replicates in a static system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also run simultaneously during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. The 24 hr EC50 value of reference substance was determined to be 0.76 mg/l. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 6.2 mg/l (95% C. I. = 3.5 to 11.2 mg/l).

 

In a supporting weight of evidence study (A. Kahru et. al., 2000), short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical. The bioassays were performed according to the standard operational procedures of the Toxkits (Daphtoxkit FTM magna). Daphtokit FTM magna follows the standard protocol as specified in the OECD TG 202 and ISO test method respecitvely. Test chemical was dissolved in deionized water. Daphnia magna (Water flea) neonates was used as a test organism. Test organism was fed with Spirulina powder. Test daphnids were exposed to test chemcial using standardized microplate test containers constructed of biologically inert materials ensure uniform exposure conditions as specified in Daphtoxkit FTM magna was used as a test vessel during the study. Study was performed under static conditions in dark for 48 hrs at 20°C. Based on the effect on mobility of the test organism, the 48 hr LC50 value was determined to be 11.2 mg/l.

 

For the test chemical, short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical (HSDB). Study was performed using Daphnia magna as a test organism under static conditions for 48 hrs. Based on the effect on mobility of the test organism, the 48 hr LC50 value was determined to be 16 mg/l.

 

Another short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical (from James Devillers, 1988 and secondary source). The study was performed following the AFNOR test method. Acetone (volume not exceeded 0.1 ml/l) was used as a dispersant solvent. Test chemical was diluted with AFNOR reconstituted hard water (pH = 7.8 to 8.2, hardness = 200 mg/l as CaCO3) for toxicity tests. Daphnia magna Straus 1820 (water flea) of < 72 h old obtained from IRCHA Laboratory was used as a test organism. Test daphnids have been cultured parthenogenetically in the Pasteur Institute Laboratory. Daphnids were cultured in 10 l tanks, with aerated hardwater (7.5 + 0.4mg/l as Ca; 5.2 + 0.3 mg/l as Mg; pH = 7). The breeding room was maintained at a regulated temperature of 22 + I°C under a photoperiod of 16 h daylight/8 h darkness. During breeding, test organism was fed with a diet of Chlorella vulgaris (1000000 cells/daphnia/h). Preliminary tests were performed in order to determine the range of concentrations at which the definitive test should be carried out. A range of geometrical concentrations were chosen so as to include chiefly the concentrations which had given 0, 10, 90, and 100% immobilization during the preliminary tests. Test daphnids (5 daphnids/vessel) were exposed to different test chemical concentrations (i.e., 0.1, 0.35, 1, 3.5, 10, 35, 100 and 350 mg/l arrange in single geometric series) in a test tube. Test vessel was covered with plastic stoppers and placed in dark conditions for 24 hrs. No aeration was provided in the test vessel during the study. During the course of the experiments, test daphnids were not fed. Control tubes (reconstituted water and dispersent-solvent) were included in the tests. Potassium dichromate was used as a reference substance during the study. All experiments were performed in triplicates. After an exposure period of 24 hr, observations of test organisms were carried out. Daphnids which were unable to swim within 15 s after stimulation by gentle agitation of the water were considered to be immobilized, even if they could still move their antennae. On the other hand; dissolved oxygen, pH, and temperature were measured at the end of the toxicity tests. The statistics, i.e., percentages of immobilization (between 10 and 90% on the basis of total number of Daphnia per concentration) were recorded and plotted as a function of concentration on log-probit paper. The points obtained were fitted to a straight line from which the IC50 was read as the abscissa of the point corresponding to 50% immobilization. The EC50 value of the reference substance was determined to be in the between range of 0.9 to 1.5 mg/l. On the basis of the effect of test chemical on mobiity of the test daphnids, the 24 hr IC50 was determined to be 13.56 mg/l with 95% C. I. of 12.338 to 14.659 mg/l.

 

In an additional study from publication (2000), short term toxicity to Thamnocephalus platyurus study was carried out for assessing the effect of test chemical. The bioassays were performed according to the standard operational procedures of the Toxkits (Thamnotoxkit FTM). Test chemical was dissolved in deionized water. Thamnocephalus platyurus larvae were exposed to test chemcial in test plate and incubated under static conditions in dark for 24 hrs at 25°C. Based on the effect on mortality of the test organism, the 24 hr LC50 value was determined to be 6 mg/l.

 

In a supporting weight of evidence study, short term toxicity to aquatic invertebrate study was carried out for 48 hrs for assessing the effect of test chemical (from peer reviewed journal and secondary sources). Ceriodaphnia dubia (Water flea) of <12 h old was used as a test organism. Test organism were acclimatized under the test conditions as reported in the main study. Each test organism was fed daily with 100 µl of yeast-trout chow -Cerophyl mixture and 50 µl of a Selenastrum capricornutum suspension. Stock solutions of the test chemical was prepared by dissolving toxicant directly into culture medium. Stock solutions were diluted to desired concentrations (4 test concentrations and 1 control) with culture medium. Test chemical concentrations were analyzed by using appropriate analytical technique atleast once during the test. Concentrations were analyzed by reverse-phase HPLC using a Water Resolve C18 column. Detection was performed on either a Hitachi F1000 fluorescence detector or a Waters 490 variable wavelength UV-Vis detector, depending on the spectral characteristics of the organic chemical. Concentration values were determined by computerized peak integration and comparison to known standards. Thus, 4 total test chemical concentrations were taken for the study. Test daphnids (10 test organism) were exposed to different concentrations of test chemical in 30 ml polystyrene cups for 48 hrs. Test conditions involve a 16:8 Light:dark photoperiod with a cool white fluorescent lamps at an intensity of 28 lux, a temperature of 25 ± 1°C, hardness of 57.07 ± 4.14 mg/l as CaCO3, pH 8.18 ± 0.04 and alkalinity of 81.00 ± 4.22 mg/l as CaCO3, respectively. All experiments were performed in a replicate. For every 24 hr over a 48 hr exposure period, mortality of the test organism was noted. 48 hr LC50 value were estimated using TOXCALC, an IBM microcomputer adaptation of a program, which calculates LC50 estimates utilizing binomial probabilities, moving averages and/or probit analysis. Survival of control animals in acute test was ≥90%. On the basis of the effect of test chemical on mortality fo the test daphnids, the 48 hr LC50 value was determined to be 3.1 mg/l.

 

For the test chemical, an acute toxicity study was conducted on aquatic invertebrates for 48 hrs for assessing the effect of test chemical (from Gerald A. LeBlanc, 1980). The study was performed using the U. S. EPA method. Daphnia magna (Water flea) of < 24 hr old obtained from laboratory stocks cultured at EG&G, Bionomics was used as a test organism. Water used to culture the organisms was deionized reconstituted well water having a total hardness of 72 + 6 mg/L as CaCO3 and a pH of 7.0 + 0.2, respectively. A stock solution of the chemical was prepared in distilled water and used to provide the desired concentrations for testing. Further, test chemical was added to 500 ml of diluted water in 2 lit jar to prepared test solution. At the initiation of test, the dissolved oxygen concentration of diluent water was > 60% of saturation. Test daphnids were exposed to test chemical in 250 ml beaker. Five daphnids were randomly placed in each 150 mL test solution within 30 minutes of the solution preparation. Test vessel was incubated for a period of 48 hrs at a temperature of 22 ± 1°C and under test conditions such as hardness of water as 72 mg/L as CaCO3, 6.7 to 8.1 and dissolved oxygen of 6.5 to 9.1 mg/l, respectively. All test experiments were performed in triplicates. A negative and positive control was run simultaneously during the study. Negative control vessel consists of the same dilution water, test conditions, and test organisms, but containing no test chemical or co-solvent. On the other hand, positive control consists of same dilution water, conditions, and a number of test organisms as in the negative control and containing the highest concentration of the co-solvent present in any test vessel. The tests were also conducted in unreplicated 500 mL solutions containing 15 daphnids if dividing the solution into triplicate test vessels presented a risk of the loss of the  test substance through volatilization or if vapors of the substance posed a high health risk to the investigators. During these tests, the dissolved oxygen concentration, pH and temperature of test solutions were measured at the initiation and termination of the toxicity tests in the high, middle and low test concentrations and controls. Dissolved oxygen concentration and temperature were measured with a YSI Model 54BP dissolved oxygen meter and combination oxygen-temperature probe. The pH's were measured with an Instrumentation Laboratory pH meter. No correction was made for control mortality. At 24 and 48 hr of an exposure period, observations of the test populations and any mortalities were recorded. The LC50's and 95% confidence limits were calculated utilizing a moving average angle method when possible. With the moving average angle method, the nominal test concentrations were transformed to logarithms and the corresponding percentage mortalities to angles. Each group of three successive angles was then averaged and the LC50 was estimated by linear interpolation between the successive concentrations whose average angles bracketed 45 °C. When test data did not meet the moving average angle method requirements, the LC50's were estimated by probit analysis by converting the concentrations to logarithms and percentage mortalities to probits and then calculating a least squares linear regression analysis. Finally, if the data did not permit a probit analysis, then a binomial probability analysis was performed on these data. Calculations were performed with a Hewlett-Packard Model 9815A calculator programmed to scan the data base. Mortality among water flea control populations never exceeded 10% in the test. On the basis of the effect of test chemical on mortality fo the test daphnids, the 24 and 48 hr LC50 value was determined to be 8.3 mg/l (95% C. I. = 5.9 to 11 mg/l) and 2.1 mg/l (95% C. I. = 1.8 to 2.5 mg/l), respectively.

On the basis of the above results, it can be concluded that the test chemical was considered to be toxic to aquatic invertebrates and hence, considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

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