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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:
2017-10-24 to 2017-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No: S7101247
CAS No: 873-75-6
EC No: 212-851-7
Aggregate State at RT: Solid
Colour: White
Molecular Weight: 187.03 g/mol
Purity: 100.0 %
Expiry Date: 31 August 2020 (minimum shelf life)
Analytical monitoring:
yes
Details on sampling:
- Sampling frequency: 4-Bromobenzyl alcohol content was determined in ISO Medium at the start and at the end of the test.
- Sampling method: Four parallel samples (5 mL per replicate) were taken from the test solutions and from the control samples.
- Sample storage conditions before analysis: The samples were prepared immediately after sampling. Formulation samples were diluted with acetonitrile and analysed by an HPLC method with UV detection. Control samples were directly injected.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. For preparation of test solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.05 g test item in 500 mL ISO medium using approx. 10 min ultrasonication. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solution.
- Differential loading: 6.25, 12.5, 25, 50 and 100 mg/L (nominal; based on the results of a non-GLP preliminary range-finding test; spacing factor = 2)
- Controls: Negative control (ISO Medium without addition of the test item), toxic reference control (with ISO Medium and the reference item Potassium dichromate; tested 05-06 September 2017)
- Evidence of undissolved material: No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Species: Daphnia magna (Straus)
- Common name: Water flea
- Age at test start: Less than 24 hours old
- Sex: Female
- Source : Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
Breeding conditions: Daphnia were bred in the Laboratory of TOXI-COOP ZRT. under similar temperature and light conditions as in the test, and in reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test. Daphnids were fed with centrifuged green alga suspension.
Acclimatisation: Test animals were bred under similar conditions as that used during the exposure period, therefore additional acclimatisation before the test was not necessary.
- Number of Animals: Twenty animals at each group, divided into 4 replicates.
- Feeding during test: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
The test temperature was in the range of 19.9 – 20.2°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.7 – 21.3°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was in the range of 7.88 – 8.07 during the test.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 7.78 – 8.25 mg/L during the test.
Salinity:
Not applicable
Conductivity:
Not measured
Nominal and measured concentrations:
Nominal concentration: 6.25, 12.5, 25, 50 and 100 mg/L
Measured mean concentration: At start of the test 5.68, 11.08, 22.94, 45.22 and 90.94 mg/L; at the end of the test 5.85, 11.42, 24.50, 47.64 and 98.25 mg/L
Thus, the measured concentrations of the test item were in the range of 89 – 92 % of the nominal at the start and 91 – 98 % at the end of the test
Details on test conditions:
TEST SYSTEM
- Test vessel: Beakers
- Type: open
- Material, fill volume: Glass, 40 mL test medium
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM: ISO Medium according to guideline


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness
- Light intensity: 611 lux

EFFECT PARAMETERS MEASURED:
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.
VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 1, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the non-GLP preliminary range-finding test (see above) five test item concentrations in a geometric series were used in the main test.
Reference substance (positive control):
yes
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
26.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
35.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- The immobilisation rate of the introduced Daphnia did not exceed 10 %. Immobility was not observed in the untreated control and no Daphnia were trapped at the water surface or showed any sign of stress.
- The dissolved oxygen concentration at the end of the test was more than 3 mg/L in control and test vessels.

All validity criteria were within acceptable limits and therefore the study was considered as valid.
Results with reference substance (positive control):
24-h EC50: 1.53 mg/L (95 % confidence limits: 1.30 – 1.78 mg/L)
Reported statistics and error estimates:
The 48-h ECx values were calculated by Probit analysis using SPSS software.
The NOEC, LOEC values were determined directly from the raw data.
Biological results are based on the mean measured test item concentrations.

Table 1:

Sampling date Nominal Mean of the measured concentrations Measured concentration in percentage of the nominal Relative standard deviation % (n=4)
24 October 2017 control not detected - -
6.25 5.68 91 0
12.5 11.08 89 0
25 22.94 92 1
50 45.22 90 1
100 90.94 91 1
26 October 2017 control not detected - -
6.25 5.85 94 2
12.5 11.42 91 2
25 24.5 98 2
50 47.64 95 2
100 98.25 98 2

Table 2: Immobilization of the test animals

Concentration
[mg/L]

Replicate

Number of
treated animals

Number of immobilised animals

24 h

48 h

Control

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

6.25

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

12.5

1

5

0

0

2

5

0

0

3

5

1

2

4

5

0

0

25

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

50

1

5

0

0

2

5

0

0

3

5

0

1

4

5

0

1

100

1

5

5

5

2

5

5

5

3

5

5

5

4

5

4

5

 

Table 3: Output of the statistical procedure for ECxestimation for immobility (Probit analysis)

Cell counts and residuals

Concentration
[mg/L]

Number of Subjects

Observed Responses

Expected Responses

Residual

Probability

6.25

5

0

.001

-.001

.000

5

0

.001

-.001

.000

5

0

.001

-.001

.000

5

0

.001

-.001

.000

12.5

5

0

.032

-.032

.006

5

0

.032

-.032

.006

5

2

.032

1.968

.006

5

0

.032

-.032

.006

25

5

0

.411

-.411

.082

5

0

.411

-.411

.082

5

0

.411

-.411

.082

5

0

.411

-.411

.082

50

5

0

1.931

-1.931

.386

5

0

1.931

-1.931

.386

5

1

1.931

-.931

.386

5

1

1.931

-.931

.386

100

5

5

3.957

1.043

.791

5

5

3.957

1.043

.791

5

5

3.957

1.043

.791

5

5

3.957

1.043

.791

Parameter estimates

Parameter

Estimate

Std. Error

Z

Sig.

95% Confidence Interval

Lower Bound

Upper Bound

Concentration

3.656

.671

5.444

.000

2.339

4.972

Intercept

-6.500

1.142

-5.693

.000

-7.642

-5.358

Chi-square Tests

Chi-Square

df

Sig.

Pearson Goodness-of-Fit Test

137.066

18

.000a

PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm)

a:Since the significance level is less than 0.050, a heterogeneity factor is used in the calculation of confidence limits.

b:Statistics based on individual cases differ from statistics based on aggregated cases.

 

Validity criteria fulfilled:
yes
Conclusions:
In this 48-hour static acute toxicity test, the obtained results showed that the test item had significant toxic effects on the mobility of Daphnia magna. The 48-h EC50 value was determined to be 60.0 mg/L (nominal). The 48-h NOEC was determined to be 50 mg/L (nominal). All validity criteria were met. The results are based on nominal test item concentrations.
Executive summary:

To evaluate the influence of the test item on the mobility and survival of Daphnia magna, an Acute Immobilisation Test according to OECD Guidleine 202, EU Method C.2 and EPA Guideline 712-C-96-114: OPPTS 850.1010 was carried out in compliance with GLP criteria. The test item (5 concentrations: 6.25, 12.5, 25, 50 and 100 mg/L in nominal) and the control were tested using 20 daphnids, divided into four groups of five animals (glass beaker; approx. 40 mL test medium) each were exposed to the test item concentrations or run as control for 48 hours in a static test. The immobility or mortality of the daphnids was determined by visual observation after 24 and 48 hours of exposure. Samples were taken from each test concentrations and from the control at the start (0 hours) and at the end of exposure (48 hours) and analysed using a HPLC-UV method. The measured concentrations of the test item were in the range of  89 – 92 % of the nominal at the start and 91 – 98 % at the end of the test. Thus, biological results are based on the nominal test item concentrations as the analytically measured test item concentrations remained within ± 20 % of the nominal over the test period of 48 hours. In result, the 48-h EC50 value was determined to be 60.0 mg/L. The 48-h NOEC was determined to be 50 mg/L. The immobilisation rate of the introduced daphnids did not exceed 10 %. Immobility was not observed in the untreated control and no daphnids were trapped at the water surface or showed any sign of stress. Furthermore, the dissolved oxygen concentration at the end of the test was more than 3 mg/L in control and test vessels. In conclusion, the obtained results showed that the test item had significant toxic effects on the mobility of Daphnia magna. All validity criteria were within acceptable limits and therefore the study is considered as valid.

Description of key information

The 48-h EC50 value of the substance was determined to be 60.0 mg/L (based on nominal concentrations). The 48-h NOEC was determined to be 50 mg/L (based on nominal concentrations).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
60 mg/L

Additional information

To evaluate the influence of the test item on the mobility and survival of Daphnia magna, an Acute Immobilisation Test according to OECD Guidleine 202, EU Method C.2 and EPA Guideline 712-C-96-114: OPPTS 850.1010 was carried out in compliance with GLP criteria. The test item (5 concentrations: 6.25, 12.5, 25, 50 and 100 mg/L in nominal) and the control were tested using 20 daphnids, divided into four groups of five animals (glass beaker; approx. 40 mL test medium) each were exposed to the test item concentrations or run as control for 48 hours in a static test. The immobility or mortality of the daphnids was determined by visual observation after 24 and 48 hours of exposure. Samples were taken from each test concentrations and from the control at the start (0 hours) and at the end of exposure (48 hours) and analysed using a HPLC-UV method. The measured concentrations of the test item were in the range of  89 – 92 % of the nominal at the start and 91 – 98 % at the end of the test. Thus, biological results are based on the nominal test item concentrations as the analytically measured test item concentrations remained within ± 20 % of the nominal over the test period of 48 hours. In result, the 48-h EC50 value was determined to be 60.0 mg/L. The 48-h NOEC was determined to be 50 mg/L. The immobilisation rate of the introduced daphnids did not exceed 10 %. Immobility was not observed in the untreated control and no daphnids were trapped at the water surface or showed any sign of stress. Furthermore, the dissolved oxygen concentration at the end of the test was more than 3 mg/L in control and test vessels. In conclusion, the obtained results showed that the test item had significant toxic effects on the mobility of Daphnia magna. All validity criteria were within acceptable limits and therefore the study is considered as valid (reference 6.1.3-1).