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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
Study period:
From July 23 to August 16, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Remarks:
validated analytical method
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted on 13 April 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Analysis of test concentrations and stability of test substance was made at the start (0 hour) on day 0 and at end of test (48 hours) for all test concentrations during range finding and main study.
15 ml of samples were collected in duplicates from each group during range finding and main study. Results are based on measured concentrations.
Vehicle:
no
Details on test solutions:
Based on the in house dissolution test, the test item was soluble in reconstituted water, therefore no vehicle was necessary for preparation of test item formulation.
Stock solution was freshly prepared prior to exposure, by weighing required quantity of test item in a beaker, a small volume of reconstituted water was added and mixed well using a glass rod. Upon dissolving test item, test formulation was transferred into a measuring cylinder and the beaker was rinsed with reconstituted water and transferred again to the measuring cylinder, rinsing process was repeated until the complete transfer of test contents. Stock solution was kept on a magnetic stirrer to maintain homogeneity. Test medium of chosen concentration was prepared by dilution of stock solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: in house maintained Daphnia magna purchased from Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Karnataka, India.
- Age of parental stock: young daphnids less than 24 hours old (derived from healthy stock) were selected for the treatment.
- Feeding during test: no feeding was done during the test.

HUSBANDARY
- Vessel: daphnids were housed in cleaned glass beakers having water holding capacity of 100 ml.
- Loading rate: 20 ml of test solution was provided for each daphnid (i.e., a volume of 100 ml for five daphnids per test vessel).
- Temperature: 20.1 to 20.9 °C
- pH: between 6.68 to 7.19
- Photoperiod: 16 hours of light and 8 hours dark cycle.
- Water: reconstituted water with pH 6.68 - 7.12, total hardness 216 mg/l(as CaCO3). The dilution water was aerated prior to use for the test so as dissolved oxygen concentration reached saturation.
- Food type: daphnids were fed with live algal cells (Pseudokirchneriella subcapitata) of 2 ml per liter during the acclimation.

ACCLIMATION
- Acclimation period: 25 gravid female daphinds were acclimatized in reconstituted water for 48 hours prior to test.
- Acclimation conditions: similar for range finding and main study.
- Type and amount of food: daphnid was fed with live algal cells at the beginning and on Day 2 of acclimation.
- Organisms selection: progenies produced on the first day are not used. Progenies produced on day two (less than 24 hours old) are transferred into the test containers and these daphnids were used for range finding as well as main study respectively.
- Conditions before range-finding test: the dissolved oxygen was between 6.64 to 6.73 mg/l, temperature of 20.1 and 20.4°C, pH of 6.67 to 6.71 and hardness of 216 mg CaCO3/l.
- Conditions before main test, the dissolved oxygen was between 6.52 to 6.71 mg/l, temperature of 20.3 and 20.5 °C, pH of 6.66 to 6.69 and hardness of 212 mg CaCO3/l.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
216 mg CaCO3/l
Test temperature:
20.1 - 20.9 °C
pH:
6.68 - 7.19
Dissolved oxygen:
7.18 - 7.54 mg/l
Nominal and measured concentrations:
0.5, 1.0, 2.4, 5.3, 11.3, 25.5 and 56.0 mg dyestuff/l, nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml glass beakers.
- Aeration: test vessels were not be aerated during the test.
- No. of organisms per vessel: 5 daphnids.
- No. of vessels per concentration: 4 replicates.
- No. of vessels per control: 4 replicate.
- No. of vessels per vehicle control (replicates):
- Biomass loading rate: 20 ml of test solution was provided for each Daphnid (i.e., a volume of 100 ml for five Daphnids per test vessel).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water, prepared in accordance with OECD guideline 202.

OTHER TEST CONDITIONS
- Adjustment of pH: no pH adjustments.
- Photoperiod: 16 hours of light and 8 hours dark cycle.

EFFECT PARAMETERS MEASURED
Each test vessel was observed for immobilized daphnids at 24 and 48 hours after the beginning of the test. Those dapnids not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. In addition to immobility, any abnormal behavior or appearance was recorded.

ENVIRONMENTAL PARAMETERS MEASUREMENT
Total hardness of the diluent water (reconstituted water) prior to its use for exposure was analyzed. Temperature, pH and dissolved oxygen were recorded at the beginning and at end of the test in control and in all the tested concentrations.

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1, 10, 50 and 100 mg dyestuff/l.
- No. of organisms per vessel: 5 daphnids.
- No. of vessels per concentration: 2 replicates.
- No. of vessels per control: 2 replicate.
- Test conditions: temperature of 20.1 and 20.7 °C, pH of 6.91 to 7.12, dissolved oxygen of 7.14 to 7.33 mg/l and hardness of 224 mg CaCO3/l.
- Results used to determine the conditions for the definitive study: after the observation period of 48 hours, immobilization percentages of 10, 50, 90 and 100 % were recorded at the nominal tested concentrations of 1, 10, 50 and 100 mg dyestuff/l, respectively.

VALIDITY CRITERIA OF THE TEST
Immobilisation in the control should not exceed 10 % and dissolved oxygen concentration at the end of the test should be ≥ 3 mg/l.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
31.72 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr. (total fraction)
Basis for effect:
mobility
Details on results:
During the main test, no clinical signs of toxicity or immobilization were observed in control and at the nominal tested concentration of 0.5 mg dyestuff/l during the 48 hour observation period.
Clinical sign of lethargy and localized on bottom of container was observed at 24 and 48 hour observation at the nominal tested concentrations of 1.0, 2.4, 5.3, 11.6 and 25.5 mg dyestuff/l.
Clinical sign of lethargy and localized on bottom of container was observed at 24 hour observation at the nominal tested concentrations of 56.0 mg dyestuff/l; all organisms resulted to be immobilized after 48 hours.
Immobilization percentages of 0, 10, 15, 30, 35, 45 and 100 % were recorded at the nominal tested concentrations of 0.5, 1.0, 2.4, 5.3, 11.6, 25.5 and 56.0 mg dyestuff/l, respectively, during the 48 hour observation period.

No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) over the 48 hour exposure period are 1.0 and 2.4 mg dyestuff/l.

MEASURED CONCENTRATIONS
During the range finding and main studies, test concentrations at 0 hour and stability at 48 hour were in the acceptable range of ± 20 % recovery to the nominal ones.

RANGE FINDING STUDY
No clinical signs of toxicity or immobilization were observed in control and at the nominal tested concentrations of 0.01 and 0.1 mg dyestuff/l during the 48 hour observation period.
Clinical sign of lethargy and localized on bottom of container was observed at 24 and 48 hour observation at the nominal tested concentrations of 1 and 10 mg dyestuff/l.
Clinical sign of lethargy and localized on bottom of container was observed at 24 hour observation at the nominal tested concentrations of 50 and 100 mg dyestuff/l, however clinical sign of lethargy continued up to 48 hour observation at the tested concentration of 50 mg dyestuff/l.
The Immobilization percentages of 10, 50, 90 and 100 % were recorded at the nominal tested concentrations of 1, 10, 50 and 100 mg dyestuff/l, respectively, during the 48 hour observation period.

MAIN TEST

Immobilization

Test Conc. (mg dyestuff/l) Replicate Initial No. of Daphnids No. of Daphnids immobilized Cummulative No.  % of Immobilisation
24 h 48 h 24 h 48 h 24 h 48 h
0 R1 5 0 0 0 0 0 0
R2 5 0 0
R3 5 0 0
R4 5 0 0
0.5 R1 5 0 0 0 0 0 0
R2 5 0 0
R3 5 0 0
R4 5 0 0
1.0 R1 5 0 1 0 2 0 10
R2 5 0 1
R3 5 0 0
R4 5 0 0
2.4 R1 5 1 0 3 3 15 15
R2 5 2 0
R3 5 0 0
R4 5 0 0
5.3 R1 5 1 1 4 6 20 30
R2 5 0 0
R3 5 1 1
R4 5 2 0

11.6

R1

5

1

0

2

7

10

35

R2

5

0

2

R3

5

0

1

R4

5

1

2

25.5

R1

5

2

0

7

9

35

45

R2

5

2

1

R3

5

2

0

R4

5

1

1

56.0

R1

5

3

2

12

20

60

100

R2

5

2

3

R3

5

4

1

R4

5

3

2

Clinical signs

Test Conc. (mg dyestuff/l)

Clinical sign at

% immobilization

24 hrs

48 hrs

0.0

No clinical signs

No clinical signs

0

0.5

No clinical signs

No clinical signs

0

1.0

Localized at bottom of container, lethargy

ocalized at bottom of container, lethargy

10

2.4

15

5.3

30
11.6 35
25.5 45
56.0 - 100

Measured concentrations

Nominal Concentration (mg dyestuff/l) Recovery (%)
0 hrs 48 hrs
0 -
0.5 114.1 80.46
1.0 100.44 86.53
2.4 98.63 104.22
5.3 95.18 86.24
11.6 106.64 94.98
25.5 99.97 83.62
56.0 85.21 81.68

RANGE-FINDING TEST

Immobilization

Test. Conc. (mg dyetuff/l) Replicate Initial No. of Daphnids No. of Daphnids immobilized Cummulative No.  % of Immobilisation
24 h 48 h 24 h 48 h 24 h 48 h
0.0 R1 5 0 0 0 0 0 0
R2 5 0 0
0.01 R1 5 0 0 0 0 0 0
R2 5 0 0
0.1 R1 5 0 0 0 0 0 0
R2 5 0 0
1 R1 5 1 0 1 1 10 10
R2 5 0 0
10 R1 5 2 1 3 5 30 50
R2 5 1 1

50

R1

5

3

1

5

9

50

90

R2

5

2

3

100

R1

5

3

2

7

10

70

100

R2

5

4

1

Clinical signs

Test Conc. (mg dyestuff/l)

Clinical sign at

% immobilization

24 hrs

48 hrs

0.0

No clinical signs

No clinical signs

0

0.01

0

0.1

0

1

Localized at bottom of container, ethargy 

Localized on bottom of container, lethargy

10

10

50

50

Lethargy

90

100

-

100

Measured concentrations

Nominal Concentration (mg dyestuff/l) Recovery (%)
0 hrs 48 hrs
0 - -
0.01 - -
0.1 - -
1 88.81 86.43
10 89.14 84.89
50 92.32 86.45
100 91.31 88.39
Validity criteria fulfilled:
yes
Remarks:
immobilisation in the control group was 0 % at termination of the test and dissolved oxygen concentration at the end of the test was 7.14 mg/l
Conclusions:
EC50 (48h): 32 mg dyestuff/l (nominal)
Executive summary:

The test item was evaluated for acute toxicity on Daphnia magna as per the OECD guideline 202. Daphnids were exposed to test item by aqueous exposure (treatment) under static condition. Daphnids were observed at 24 and 48 hours for signs of toxicity and immobilisation.

Based on the results of the range finding study, main study was conducted at test concentrations of 0.5, 1.0, 2.4, 5.3, 11.6, 25.5 and 56 mg dyestuff/l of test medium along with control.

No clinical signs of toxicity were observed in control and at 0.5 mg dyestuff/l during the 48 hour observation period.

Clinical signs of localized on bottom of container and lethargy was observed at the nominal tested concentrations of 1.0, 2.4, 5.3, 11.6 and 25.5 mg dyestuff/l during the 24 and 48 hour observation period. Clinical signs of localized on bottom of container and lethargy was observed at the nominal tested concentration of 56.0 mg dyestuff/l during the 24 hour observation period; all organisms resulted to be immobilized after 48 hours.

No immobilization was observed in control and at 0.5 mg dyestuff/l during the 48 hour observation period. Immobilization percentages of 10, 15, 30, 35, 45 and 100 % were recorded at concentrations of 1.0, 2.4, 5.3, 11.6, 55.5 and 56.0 mg dyestuff/l, respectively, during the 48 hour observation period.

Based on test results, it is concluded that the 48 hour acute median effective concentration (EC50) is 31.72 mg dyestuff/l; the 95 % confidence interval with lower limit of 17.46 mg dyestuff/l and upper limit of 57.63 mg/l. No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) over the 48 hour exposure period are 1.0 and 2.4 mg dyestuff/l.

Samples from all the test concentrations were collected at 0 hour and at 48 hour analyzed for the test concentrations and stability. Test concentrations at 0 hour and stability at 48 hour were in the acceptable range of ± 20 % recovery of nominal concentrations.

Conclusion

EC50 (48h): 32 mg dyestuff/l (nominal)

Description of key information

EC50 (48h): 32 mg dyestuff/l (nominal)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
32 mg/L

Additional information

A study from 1986 on the short-term toxicity potential of Acid Blue 193 to invertebrates was available. Unfotunately, it was not possible to retreive the detailed composition of test sample, thus it cannot be confirmed if the composition was comparable to those currently placed on the market; it's only known that it was a concentrated sample.

The study was run on Daphnia magna following OECD guideline 202, part I (1981). Under test conditions, the substance resulted to be very slightly toxic to daphnids; the EC50 value after the exposure period of 24-hours resulted to be higher than 100 mg/l (nominal).

Due to the uncertainty related to the composition tested, a new test was run. The test item was evaluated for acute toxicity on Daphnia magna as per OECD guideline 202. Daphnids were exposed to test item by aqueous exposure (treatment) under static condition. Daphnids were observed at 24 and 48 hours for signs of toxicity and immobilisation.

Based on the results of the range finding study, main study was conducted at nominal concentrations of 0.5, 1.0, 2.4, 5.3, 11.6, 25.5 and 56 mg dyestuff/l of test medium along with control.

No clinical signs of toxicity were observed in control and at 0.5 mg dyestuff/l during the 48 hour observation period.

Clinical signs of localized on bottom of container and lethargy were observed at concentrations of 1.0, 2.4, 5.3, 11.6 and 25.5 mg dyestuff/l during the 24 and 48 hour observation period. Clinical signs of localized on bottom of container and lethargy was observed at 56.0 mg dyestuff/l during the 24 hour observation period; all organisms resulted to be immobilized after 48 hours.

No immobilization was observed in control and at 0.5 mg dyestuff/l during the 48 hour observation period. The Immobilization percentages of 10, 15, 30, 35, 45 and 100 % were recorded at 1.0, 2.4, 5.3, 11.6, 55.5 and 56.0 mg dyestuff/l, respectively, during the 48 hour observation period. Based on the results, it is concluded that the 48 hour acute median effective concentration (EC50) is 31.72 mg dyestuff/l; the 95 % confidence interval with lower limit of 17.46 mg dyestuff/l and upper limit of 57.63 mg/l. No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) over the 48 hour exposure period are 1.0 and 2.4 mg dyestuff/l.

Samples from all the test concentrations were collected at 0 hour and at 48 hour analyzed for the test concentrations and stability. Test concentrations and stability were in the acceptable range of ± 20 % of the nominal values.