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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March 2015 - 25 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were performed at the applied test concentration levels and control at the beginning and at the end of the experiment. The samples were analysed by an HPLC-UV method.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a saturated test item stock solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) and stirred for about an hour using magnetic stirrer. The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the experiment) .
- Evidence of undissolved material (e.g. precipitate, surface film, etc): the non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the 100 % saturated solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: National Institute of Public Health, 1097 Budapest Gyáli u. 2-6. Hungary
- Age at study initiation (mean and range, SD): < 24 h old.
- Feeding during test: no.

ACCLIMATION
There was no acclimatization because the water used was similar to the culture water.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
247 mg/L (as CaCO3)
Test temperature:
19.9 – 20.2 °C
pH:
6.83 – 7.04
Dissolved oxygen:
8.0 – 8.9 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0, 0.205, 0.512, 1.28, 3.2, 8 and 20 % saturated solution.
Measured concentrations: 0.0, 0.0007, 0.0025, 0.017, 0.046, 0.118 and 0.328 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stopper with a volume of 50 mL
- Type (delete if not applicable): closed
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO medium, according to OECD 202)
- Culture medium different from test medium: no.
- Intervals of water quality measurement: the water temperature was measured daily in each test vessel. The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the test.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): the immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: two concentration range-finding tests were conducted. Taking into account that the test item is a highly volatile substance, the second test was performed with confirmatory analytical measurements at the start and at the end of the experiment in order to better define loss of the test substance during the exposure period.
- Test concentrations:
First Preliminary Range-finding test: 0, 0.1, 1, 10 and 100% saturated solution (open vessels; 10 daphnids/concentration; 2 replicates; 48 h under static conditions).
Second Preliminary Range-finding test: 0, 0.512, 3.2 and 20% saturated solution (closed vessels; 15 daphnids/concentration; 3 replicates; 48 h under static conditions).
- Results used to determine the conditions for the definitive study: because significant immobility was observed during the performed preliminary range-finding tests and taking into account that the lowest concentration tested should preferably give no effect in the definitive test, therefore a wide concentration range was used and six test concentrations in a geometric series (factor 2.5) and one control were used in the main test under static conditions. The following nominal concentrations were tested: 0.205, 0.512, 1.28, 3.2, 8 and 20 % saturated solution.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (03-04 March 2015)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95 % CL: 0.024 – 0.039 mg/L)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.017 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.046 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Statistically significantly different number of immobilised animals compared to the control was observed at concentrations of 0.0460 mg/L after 48 hours of exposure and above (measured concentration). Detailed data regarding the number and percentage of immobilised animals after 24 and 48 h of exposure is shown in Table 1. Other adverse effects could not be observed during the experiment.
Results with reference substance (positive control):
24h EC50: 1.05 mg/L, (95 % confidence limits: 0.89 – 1.23 mg/L)
Reported statistics and error estimates:
The 24 and 48 hours EC50 values were determined by Probit analysis with 95 % confidence limits using TOXSTAT software. For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software. The 48 hours EC100 values were determined directly from the raw data.

Endpoint values:

24h- EC50value: 0.090 mg/L (95 % confidence limits: 0.071 – 0.114 mg/L)

48h- EC50value: 0.030 mg/L(95 % confidence limits: 0.024 – 0.039 mg/L)

48h- EC100value: 0.328 mg/L

48h- No-Observed Effect Concentration (NOEC): 0.017 mg/L

48h- Lowest Observed Effect Concentration (LOEC): 0.046 mg/L

Table 1. Number and percentage of immobilised animals - definitive test.

Concentration

Number of treated animals

Immobilised animals

Nominal

(% sat.sol.)

Calculated or Measured

[mg/L]

24 hours

48 hours

number

%

number

%

Control

0.0

20

0

0

0

0

0.205

0.0007

20

0

0

1

5

0.512

0.0025

20

3

15

4

20

1.28

0.0172

20

1

5

6

30

3.2

0.0460

20

1

5

9*

45

8

0.118

20

11*

55

14*

70

20

0.328

20

20*

100

20*

100

*: statistically significantly different compared to the control values (Dunnett’s Test; α= 0.05).

Table 2. Calculation of exposure concentrations.

Nominal
concentration
(
% sat. sol.)

Measured concentration
start (mg/L)

Measured concentration
end (mg/L)

Geometric mean

Extrapolated value

mg/L

Control

nd

nd

-

-

0.205

-*

-*

-

0.00067

0.512

0.0025**

0.0025**

0.0025

-

1.28

0.0184

0.0161

0.0172

-

3.2

0.0505

0.0419

0.0460

-

8

0.123

0.113

0.118

-

20

0.341

0.316

0.328

-

n.d. – not detected

*concentration was not measured due to the extremely low concentration

**concentration was below the quantification limit. In order to calculate mean value, concentration was taken as the half of the Limit of Quantification (LOQ = 0.005 mg/L) according to OECD 23; paragraph 3.3.

The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item. The biological results are based on the calculated and measured geometric mean concentrations.

Validity criteria fulfilled:
yes
Remarks:
(there were no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L).
Conclusions:
The EC50 (based on immobilisation) of test item was determined to be 0.030 mg/L in Daphnia magna after 48 hours of exposure.
Executive summary:

The acute toxicity of the test substance on Daphnia magna was assessed in an Acute immobilisation test over an exposure period of 48 hours in a static, closed system. The study was performed in accordance with the OECD Guideline 202 and following the Principles of GLP. Based on the results of the performed preliminary range-finding tests, six test concentrations in a geometric series with a separation factor of 2.5 and one untreated control were tested in the main experiment.The nominal concentrations of test item used in the main experiment were: 0.205, 0.512, 1.28, 3.2, 8 and 20 % saturated solutions. Analytical measurements were performed at the beginning and at the end of the experiment.The corresponding calculated and measured geometric mean test item concentrations were: 0.0007, 0.0025, 0.017, 0.046, 1.118, 0.328 mg/L.The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item.The biological results are based on the calculated and measured geometric mean concentrations. Twenty animals, divided into four groups (Erlenmeyer flasks with stopper) of five animals each were used at the test and for the control groups. After 48 hours of exposure, the EC50 based on immobilisation was determined to be 0.030 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance 7-chloro-3-methyl-1-benzothiophene (SER-4) which shares the aryl functional group chlorophenyl with the substance 1-[(4-chlorophenyl)sulfanyl]propan-2-one (SER-3) also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.033 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mobility
Validity criteria fulfilled:
yes
Conclusions:
Based on the available information for the read-across approach, the 48h-EC50 of the test item in Daphnia magna is 0.033 mg/L.

Description of key information

Read-across from analogue substance: Key study: Test method according to OECD Guideline 202. GLP study. The EC50 (based on immobilisation) of test item was determined to be 0.030 mg/L in Daphnia magna after 48 hours of exposure. Based on the available information for the read-across approach, the 48h-EC50 of the test item in Daphnia magna is 0.033 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.03 mg/L

Additional information

Read-across from analogue substance: Key study: The acute toxicity of the test substance on Daphnia magna was assessed in an Acute immobilisation test over an exposure period of 48 hours in a static, closed system. The study was performed in accordance with the OECD Guideline 202 (GLP study). Based on the results of the performed preliminary range-finding tests, six test concentrations in a geometric series with a separation factor of 2.5 and one untreated control were tested in the main experiment.The nominal concentrations oftest item used in the main experiment were: 0.205, 0.512, 1.28, 3.2, 8 and 20 % saturated solutions. The corresponding calculated and measured geometric mean test item concentrations were: 0.0007, 0.0025, 0.017, 0.046, 1.118, 0.328 mg/L.The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item.The biological results are based on the calculated and measured geometric mean concentrations. Twenty animals, divided into four groups (Erlenmeyer flasks with stopper) of five animals each were used at the test and for the control groups. After 48 hours of exposure, the EC50 based on immobilisation was determined to be 0.030 mg/L. Based on the available information for the read-across approach, the 48h-EC50 of the test item in Daphnia magna is 0.033 mg/L.