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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:
14 Aug 2017 - 23 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10, 18, 32, 56, 100, 180 mg/L (2nd Full Test)
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control, 2.0 mL from the approximate centre of the test vessels, at t=0 h and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L (in the combined limit/range finding test and 1st full test) and 180 mg/L (in the 2nd full test) applying approximately 20 minutes of magnetic stirring followed by approximately 20 minutes of ultrasonic waves to accelerate dissolution of the test item in medium. For the two full tests, these treatments were sufficient to fully dissolve the test item in test media. For the combined limit/range-finding test, undissolved particles were still observed in the test solution. Consequently, the highest test concentration was collected by means of siphoning after an additional treatment of approximately 20 minutes with ultrasonic waves followed by 14 minutes of magnetic stirring. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age at start of test: < 24 hours, from parental daphnids of more than two weeks old
- Feeding during test: no

CULTIVATION
- Start of batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: after 7 days of cultivation half of the medium twice a week
- Temperature of the medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae
- Medium: M7 medium
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L (CaCO3)
Test temperature:
20°C
pH:
At start: 7.9
At end: 8.2
Dissolved oxygen:
At start: 8.9-9.1 mg/L
At end: 8.7-8.8 mg/L
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56, 100, 180 mg/L
Measured: 11, 20, 35, 62, 109, 195 mg/L (average exposure concentration, see table in 'Any other information on results' for Measured concentrations in all samples. Since the test item showed a steep dose-response curve at concentration slightly higher than 100 mg/L, it was decided to base the effects parameters on the average exposure concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL all-glass, containing 50 mL test solution
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: within one hour after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, made with tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes, M7 medium
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control; temperature of the medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 hours and at 48 hours.

RANGE-FINDING STUDY
- Test concentrations: Control, 0.1, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (performed Sep 2017)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
107 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 91-127 mg/L
Details on results:
In a first Full test, no immobility was observed in the control and at any of the concentrations tested during the 48 hours of exposure, which was unexpected based on the results of the combined limit/range-finding test. The test item appeared to have a steep dose-response curve starting at concentrations slightly higher than 100 mg/L. In fact, effects were complete at an average exposure concentration of 110 mg/L in the combined limit/range-finding test, while no effects were observed at an average exposure concentration of 102 mg/L in the first full test. In order to demonstrate this and to determine the effects parameters, it was decided to perform a second full test with an additional test concentration of 180 mg/L.

In the second full test, no immobilisation was observed in the control and at the four lowest test concentrations throughout the test duration. At the two highest test concentrations, 62 and 77% of the daphnids were immobile after 24 hours of exposure. The observed effects increased to 71 and 86% at the end of the test. Results from the second full test were used to calculate effect parameters.

Six daphnids instead of five were added to vessel B at concentration of 100 mg/L and vessel A and D at concentration of 180 mg/L. This was taken in consideration during calculation of the effects parameters.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.
- The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
Reported statistics and error estimates:
The 24 and 48h-EC50-value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding average measured test item concentrations using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

 Table 4: Second Final Test: Test Samples

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

17 Oct 2017

23 Oct 2017

0

n.d.

n.a.

 

 

 

 

10

10.6

106

 

 

 

 

18

19.6

109

 

 

 

 

32

35.1

110

 

 

 

 

56

61.6

110

 

 

 

 

100

110

110

 

 

 

 

180

197

110

 

48

19 Oct 2017

23 Oct 2017

0

n.d.

n.a.

n.a.

 

 

 

10

10.6

106

100

 

 

 

18

19.6

109

100

 

 

 

32

35.4

111

101

 

 

 

56

61.6

110

100

 

 

 

100

109

109

99

 

 

 

180

193

107

98

1     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

n.d.Not detected.

n.a.Not applicable.

 

Table5: Number of Introduced Daphnids and Incidence of Immobility in the 2ndFull Test

Time (h)

Replicate

Ethanol, 2,2’-[(3-methylphenyl)imino]bis-;

Average exposure concentration (mg/L)

Control

11

20

35

62

109

195

0

A

5

5

5

5

5

5

6*

B

5

5

5

5

5

6*

5

C

5

5

5

5

5

5

5

D

5

5

5

5

5

5

6*

Total introduced

20

20

20

20

20

21

22

 

24

A

0

0

0

0

0

2

5

B

0

0

0

0

0

4

2

C

0(2)

0

0

0

0

3

4

D

0

0

0

0

0

4

6

Total immobilised

0

0

0

0

0

13

17

Effect %

0

0

0

0

0

62

77

 

 

 

 

 

 

 

48

A

0

0

0

0

0

2

5

B

0

0

0

0

0

6

3

C

0

0

0

0

0

2

5

D

0

0

0

0

0

5

6

Total immobilised

0

0

0

0

0

15

19

Effect %

0

0

0

0

0

71

86

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

* Six instead of five daphnids were introduced to the test solution

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria, see 'Overall remarks' section.
Conclusions:
The 48h-EC50 for Daphnia magna exposed to Ethanol, 2,2’-[(3-methylphenyl)]-bis- was 107 mg/L based on average exposure concentrations (95% confidence interval between 91 and 127 mg/L).
Executive summary:

In a static 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 10, 18, 32, 56, 100 and 180 mg/L, 5 daphnids per vessel and 4 replicates per concentration. Average exposure concentrations were measured to be 11, 20, 35, 62, 109 and 195 mg/L. The effect parameters were based on the average exposure concentrations. No immobilisation was observed in the control and at the four lowest test concentrations throughout the test duration. At the two highest test concentrations, 62 and 77% of the daphnids were immobile after 24 hours of exposure. The observed effects increased to 71 and 86% at the end of the test. A steep dose-response curve for immobility was observed at concentration slightly higher than 100 mg/L. The 48h-EC50 for Daphnia magna exposed to the test item was 107 mg/L (95% confidence interval 91 -127 mg/L). The study met all validity criteria and is considered to be reliable without restrictions.

Description of key information

In a study according to OECD 202 the 48h-EC50 for Daphnids was determined to be 107 mg/L, based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
107 mg/L

Additional information

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 10, 18, 32, 56, 100 and 180 mg/L, 5 daphnids per vessel and 4 replicates per concentration. Average exposure concentrations were measured to be 11, 20, 35, 62, 109 and 195 mg/L. The effect parameters were based on the average exposure concentrations. No immobilisation was observed in the control and at the four lowest test concentrations throughout the test duration. At the two highest test concentrations, 62 and 77% of the daphnids were immobile after 24 hours of exposure. The observed effects increased to 71 and 86% at the end of the test. A steep dose-response curve for immobility was observed at concentration slightly higher than 100 mg/L. The 48h-EC50 for Daphnia magna exposed to the test item was 107 mg/L (95% confidence interval 91 -127 mg/L).