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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:
08 Jun 2018 - 18 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and control
- Sampling method: 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 a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle for a period of three hours. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS 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 of parental stock: > 2 weeks
- Age at test initiation: <24 hours
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

CULTIVATION
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of M7 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 medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
19 - 20°C
pH:
At t=0 h: 8.0 - 8.1
At t=48 h: 8.2
Dissolved oxygen:
At t=0 h: 8.0 mg/L
At t=48 h: 9.1-9.2 mg/L
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56, 100% of Saturated Solution prepared at 100 mg/L
Average measured exposure concentration: 0.070, 0.15, 0.22, 0.41, 0.82 mg/L. At the end of the test, the concentrations were at 64-104% relative to the initial concentrations. The average exposure concentrations were calculated and used to determine the effect parameters. See 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- Feeding: no feeding
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium prepared with tap water purified by Reverse Osmosis and the following salts: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Culture medium different from test medium: Yes
- 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 medium: continuously in a temperature control vessel, beginning at the start of the test.

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

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

RANGE-FINDING STUDY
- Combined Limit/Range-Finding Test concentrations: 1.0, 10, 100% of SS prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, expected EC50 between 10 and 100% SS.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (performed March 2018)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 0.18 - 0.24 mg/L
Details on results:
- Behavioural abnormalities: none observed in the Control group.
- Mortality of control: none
- No immobility was observed in the control and the lowest test concentration throughout the exposure period while at the end of the test, a dose-related increase of immobility was observed at average concentrations of 0.15 mg/L and higher, reaching 100% immobility at the two highest test concentrations. See Table 2 in 'Any other information on results' for full details on immobility.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid?
Yes
- The 24h-EC50 was 0.80 mg/L with a 95% confidence interval between 0.71 and 0.90 mg/L.
- The 48h-EC50 was 0.33 mg/L with a 95% confidence interval between 0.29 and 0.38 mg/L.
- The 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 the Test Facility.
Reported statistics and error estimates:
The 24 and 48h-EC50-values were calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (average) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Average Exposure Concentrations 

4,4’-methylenebis-N-sec-butylaniline

%SS prep. at 100 mg/L

Measured concentrations (mg/L)

Average exposure conc. (mg/L)

t=0h

t=48h

10

0.0881

0.0563

0.070

18

0.173

0.126

0.15

32

0.220

0.229

0.22

56

0.425

0.397

0.41

100

0.924

0.733

0.82

Table 2 Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)

Replicate

4,4’-methylenebis-N-sec-butylaniline; Average conc. (mg/L)

Control

0.070

0.15

0.22

0.41

0.82

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

3

5

B

0

0

0

0

3

5

C

0

0

0

0

2

5

D

0

0

0

0

1

5

Total immobilised

0

0

0

0

9

20

Effect %

0

0

0

0

45

100

 

 

 

 

 

 

48

A

0

0

0

4

5

5

B

0

0

1

2

5

5

C

0

0

1

4

5

5

D

0

0

1

2

5

5

Total immobilised

0

0

3

12

20

20

Effect %

0

0

15

60

100

100

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 48h-EC50 for Daphnia magna exposed to the test substance was 0.21 mg/L based on average exposure concentrations (95% confidence interval between 0.18 and 0.24 mg/L).
Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Saturated Solutions of the test substance prepared at a loading rate of 100 mg/L (10, 18, 32, 56 and 100% of SS) and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). At the end of the test, measured concentrations were at 64-104% relative to initial. Therefore, geometric mean measured exposure concentrations were used to determine the effect parameters. No immobility was observed in the control and the lowest test concentration throughout the exposure period while at the end of the test, a dose-related increase of immobility was observed at average concentrations of 0.15 mg/L and higher, reaching 100% immobility at the two highest test concentrations. The 48h-EC50 for immobility was 0.21 mg/L based on geometric mean measured exposure concentrations (95% confidence interval between 0.18 and 0.24 mg/L). The study is considered to be reliable without restrictions.

Description of key information

48h-EC50 (immobility): 0.21 mg/L (geometric mean measured exposure concentration)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.21 mg/L

Additional information

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Saturated Solutions of the test substance prepared at a loading rate of 100 mg/L (10, 18, 32, 56 and 100% of SS) and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). At the end of the test, measured concentrations were at 64-104% relative to initial. Therefore, geometric mean measured exposure concentrations were used to determine the effect parameters. No immobility was observed in the control and the lowest test concentration throughout the exposure period while at the end of the test, a dose-related increase of immobility was observed at average concentrations of 0.15 mg/L and higher, reaching 100% immobility at the two highest test concentrations. The 48h-EC50 for immobility was 0.21 mg/L based on geometric mean measured exposure concentrations (95% confidence interval between 0.18 and 0.24 mg/L). The study is considered to be reliable without restrictions.