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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mg/l
- Sampling method: The stock solution was prepared by dissolving 500 mg of test chemical in 500 ml of M7 medium with 24 hours of continous stirring to get the final concentration of 1000 mg/L, followed by analytical determination. The final solubility value obtained after analytical detection was 16.06 mg/L. The remaining test solutions were prepared by dilution from the above stock solution.
- Sample storage conditions before analysis: sample was analysed immediately after preperation
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: 500 mg of test item was transferred and made up to 500 mL in a volumetric flask using M7 medium (Stock solution). From the stock solution 0 (control), 0.625, 1.25, 2.5, 5 and 10 mg/L were prepared using M7 medium in a glass test vessel.
- Controls: M7 Medium (Control),
- 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
- Source: Daphnia magna were obtained from Denmark Technical University and maintained at Unique Ecotox Research Laboratory, Nagpur.
- Age at study initiation (mean and range, SD): <24 hr
- Length at study initiation (length definition, mean, range and SD): 0.4 cm
- Feeding during test : No
Others: A population of Daphnia magna of synchronized age structure has been maintained for more than 2 months in the test facility under constant temperature conditions (18 to 22°C) at a 16 : 8 hour light-dark photoperiod (illumination: > 1000 lux). The culture media (M7 medium') was partly renewed twice a week. During the maintenance of test organism, test daphnids were fed with unicellular green algae (Selenestrum capricornutum).

ACCLIMATION
- Acclimation period: 48 hours prior test
- Acclimation conditions (same as test or not): Yes (Test daphnids were acclimatized)
- Type and amount of food: Food was provided during acclimatization.
- Health during acclimation (any mortality observed): healthy no mortality was observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
>140 mg CaCO3/L
Test temperature:
20 °C
pH:
Control 7.2 - 7.0
Dissolved oxygen:
7.1-7.4 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.625, 1.25, 2.5, 5 and 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 mL Beakers
- Type (delete if not applicable): open / closed Loosely covered with glass lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glass, volume 20 mL
- Volume of solution: 20 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 5 daphnids/20 mL test medium

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 1337-1346 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation
VEHICLE CONTROL PERFORMED: Not applicable
- Spacing factor for test concentrations:2

RANGE-FINDING STUDY
Based on the available data on the test chemical range finding test was not conducted directly main study was intaited to draw the conclusion on test substance toxicity.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.671 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: No mortality in control
- Other adverse effects control: None
- Immobilisation of control: No immobilisation
- Abnormal responses: None in control
Results with reference substance (positive control):
- Results with reference substance valid? Yes valid
EC50: 0.650 mg/L

Lower limit = 0.631 mg/L
Reported statistics and error estimates:
48h EC50 value were calculated by probit analysis using STAT Plus Software, version 8.
Concentration After Analytical Determination
 
 

0 Hour

0 Hour

48 Hours

48 hours

Sr. No

Concentration’s(mg/L)

Absorbance (mean)

Analytical concentrations

Absorbance (mean)

Analytical concentrations

1 control 00 00 00 00
2 0.625 0.05 0.51 0.05 0.53
3 1.25 0.10 0.98 0.09 0.92
4 2.5 0.22 2.21 - -
5 5 0.47 4.62 - -
6 10 0.89 8.84 - -
 
Conclusion:

The test concentrations were measured and found to remainwithin in ±20%of nominal. Hence, the EC50(0.671 mg/L)was expressed based onnominalconcentrations.

Result tables -
Nominal conc. (mg/l) Number of Daphnids/replicate Immobility
0h 24h 48h Cumulative Immobilization Percent Inhibition
control 5 R1=0 R2=0 R3=0 R4=0 R1=0 R2=0 R3=0 R4=0 R1=0 R2=0 R3=0 R4=0 0 0
0.625 5 R1=0 R2=0 R3=0 R4=0 R1=0 R2=0 R3=0 R4=0 R1=2 R22= R3=2 R4=1 7 35
1.25 5 R1=0 R2=0 R3=0 R4=0 R1=5 R2=4 R3=5 R4=4 R1=0 R2=0 R3=1 R4=0 19 95
2.5 5 R1=0 R2=0 R3=0 R4=0 R1=5 R2=5 R3=5 R4=5 R1=5 R2=5 R3=5 R4=5 20 100
5 5 R1=0 R2=0 R3=0 R4=0 R1=5 R2=5 R3=5 R4=5 R1=5 R2=5 R3=5 R4=5 20 100
10 5 R1=0 R2=0 R3=0 R4=0 R1=5 R2=5 R3=5 R4=5 R1=5 R2=5 R3=5 R4=5 20 100
Any abnormal behavior(if any) Actively moving Mortalities recorded in highest concentrations Slow movement as compared to control    

Table - Temperature, DO and pH measurements

0 Hour
Nominal conc. (mg/l) Temperature(° C) DO(mg/l) pH
control R1=21.2 R2=21.2 R3=21.2 R4=21.2 R1=7.1 R2=7.1 R3=7.1 R4=7.1 R1=7.6 R2=7.6 R3=7.6 R4=7.6
0.625 R1=21.2 R2=21.2 R3=21.2 R4=21.2 R1=7.4 R2=7.4 R3=7.4 R4=7.4 R1=7.1 R2=7.1 R3=7.1 R4=7.1
1.25 R1=21.2 R2=21.2 R3=21.2 R4=21.2 R1=7.1 R2=7.1 R3=7.1 R4=7.1 R1=7.2 R2=7.2 R3=7.2 R4=7.2
2.5 R1=21.2 R2=21.2 R3=21.2 R4=21.2 R1=7.1 R2=7.1 R3=7.1 R4=7.1 R1=7.3 R2=7.3 R3=7.3 R4=7.3
5 R1=21.5 R2=21.5 R3=21.5 R4=21.5 R1=7.2 R2=7.2 R3=7.2 R4=7.2 R1=7.3 R2=7.3 R3=7.3 R4=7.3
10 R1=21.5 R2=21.5 R3=21.5 R4=21.5 R1=7.1 R2=7.1 R3=7.1 R4=7.1 R1=7.3 R2=7.3 R3=7.3 R4=7.3
24 Hours
Nominal conc. (mg/l) Temperature(° C) DO(mg/l) pH
control R1=20.4 R2=20.4 R3=20.4 R4=20.4 R1=6.8 R2=6.8 R3=6.8 R4=6.8 R1=7.2 R2=7.2 R3=7.2 R4=7.2
0.625 R1=20.4 R2=20.4 R3=20.4 R4=20.4 R1=6.6 R2=6.6 R3=6.6 R4=6.6 R1=7.1 R2=7.1 R3=7.1 R4=7.1
1.25 R1=20.4 R2=20.4 R3=20.4 R4=20.4 R1=6.4 R2=6.4 R3=6.4 R4=6.4 R1=7.1 R2=7.1 R3=7.1 R4=7.1
2.5 R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=-
5 R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=-
10 R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=-
48 hours
Nominal conc. (mg/l) Temperature(° C) DO(mg/l) pH
control R1=21.3 R2=21.3 R3=21.3 R4=21.3 R1=6.1 R2=6.1 R3=6.1 R4=6.1 R1=7.0 R2=7.0 R3=7.0 R4=7.0
0.625 R1=21.3 R2=21.3 R3=21.3 R4=21.3 R1=6.0 R2=6.0 R3=6.0 R4=6.0 R1=7.3 R2=7.3 R3=7.3 R4=7.3
1.25 R1=21.3 R2=21.3 R3=21.3 R4=21.3 R1=6.2 R2=6.2 R3=6.2 R4=6.2 R1=7.4 R2=7.4 R3=7.4 R4=7.4
2.5 R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=-
5 R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=-
10 R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=- R1=- R2=- R3=- R4=-
 
Validity criteria fulfilled:
yes
Remarks:
In the control, there was no daphnid found immobilized or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was ≥ 3 mg/ L in control and test vessels
Conclusions:
Based on the effect on mobility of test daphnids, the 48-h EC50 of test chemical to daphnid, Daphnia magna was reported to be 0.671 mg/L.
Executive summary:

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The test daphnids were acclimatized 48 hours prior to the test chemical exposure. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test chemical formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (using a spacing factor of 2) was conducted using 0 (control), 0.625, 1.25, 2.5, 5 and 10 mg/L mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and after 48 hours of exposure, which were found in acceptable range. Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.2 -7.6), temperature (20-21°C), dissolve oxygen (7.1 - 7.4 mg/L), hardness (>140 mg CaCO3/L), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria. Feed was not provided during the test. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups but 35%, 95%, 100%, 100% and 100% immobilisation were observed in the test concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/L, respectively. The 48-h EC50 of test chemical to daphnid, Daphnia magna are 0.671 mg/l. The 48-h EC50 of reference item (Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.65 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Thus, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in 'aquatic acute/chronic category 1' as per the CLP classification criteria.

Description of key information

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The test daphnids were acclimatized 48 hours prior to the test chemical exposure. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test chemical formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (using a spacing factor of 2) was conducted using 0 (control), 0.625, 1.25, 2.5, 5 and 10 mg/L mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and after 48 hours of exposure, which were found in acceptable range. Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.2 -7.6), temperature (20-21°C), dissolve oxygen (7.1 - 7.4 mg/L), hardness (>140 mg CaCO3/L), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria. Feed was not provided during the test. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups but 35%, 95%, 100%, 100% and 100% immobilisation were observed in the test concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/L, respectively. The 48-h EC50 of test chemical to daphnid, Daphnia magna are 0.671 mg/l. The 48-h EC50 of reference item (Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.65 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Thus, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in 'aquatic acute/chronic category 1' as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.671 mg/L

Additional information

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The test daphnids were acclimatized 48 hours prior to the test chemical exposure. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test chemical formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (using a spacing factor of 2) was conducted using 0 (control), 0.625, 1.25, 2.5, 5 and 10 mg/L mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and after 48 hours of exposure, which were found in acceptable range. Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.2 -7.6), temperature (20-21°C), dissolve oxygen (7.1 - 7.4 mg/L), hardness (>140 mg CaCO3/L), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria. Feed was not provided during the test. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups but 35%, 95%, 100%, 100% and 100% immobilisation were observed in the test concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/L, respectively. The 48-h EC50 of test chemical to daphnid, Daphnia magna are 0.671 mg/l. The 48-h EC50 of reference item (Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.65 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Thus, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be classified in 'aquatic acute/chronic category 1' as per the CLP classification criteria.