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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:
March 18, 2020 - March 20, 2020.
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)
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 no. 23
Version / remarks:
2019
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)
Analytical monitoring:
no
Remarks:
Not feasible, see attached background material.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Saturated test item solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the appropriate 100% saturated solution. The maximum amount of test material in the medium was exposed applying this procedure. This WAF method is considered to be appropriate to maximize the amount of dissolved test item in the media. The test solutions were prepared just before introduction of the Daphnia (start of the treatments).
- Eluate: dilution water (ISO-medium)
- Controls: untreated control consisted of ISO-medium without addition of test item; reference control was performed with potassium dichromate.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnid
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Age at study initiation (mean and range, SD): less than 24 h old
- Method of breeding: bred in Ecotoxicological Laboratory of Charles River Laboratories Hungary Kft. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour.
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary.
- Feeding during test : no

ACCLIMATION
- Acclimation period: No, culture conditions same as test.
- Health during acclimation (any mortality observed): 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 were used for the study and there was no delay in the production of the first brood.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
244 mg/L as CaCO3
Test temperature:
20.4 – 20.8°C
pH:
7.82 – 7.95
Dissolved oxygen:
8.4 – 9.0 mg/L.
Nominal and measured concentrations:
Nominal concentrations: 0.064, 0.16, 0.4, 1.0 and 2.5 % saturated solutions (of 100 mg/L loading rate)
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: all-glass beakers, containing at least 5 mL test solution/animal.
- Volume of solution: 25 mL
- Aeration: no
- Renewal rate of test solution: 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. Separate stock solutions of individual trace elements were first prepared in deionised water. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution).
- Culture medium different from test medium: no

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (including mortality), at 24 hours and at 48 hours for all concentrations; pH an dissolved oxygen, at the beginning and at the end of the test, for all loading rates and the control; and temperature of medium, at the beginning at the start of the test, plus continuously in a temperature control vessel.

RANGE-FINDING STUDY
- A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions. During the formulation procedure the stock solution was prepared as in main test.
- Test concentrations: 0, 0.1, 1.0, 10.0, and 100.0% of saturated solution (nominal loading rate of 100.0 mg/L).
- Results used to determine the conditions for the definitive study: Because significant immobility was observed at the concentration levels of 1.0, 10.0 and 100.0 % saturated solutions during the preliminary range-finding test 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, five test concentrations in a geometric series with a separation factor of 2.5 and one untreated control were tested in the main experiment under semi-static conditions. The nominal concentrations of test item used in the main experiment were 0.064, 0.16, 0.4, 1.0 and 2.5 % saturated solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (batch no.: A0345704)
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
0.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
- Observations on body length and weight: none.
- Other biological observations: none.
- Mortality of control: no.
- Other adverse effects control: no.
- Immobilisation of control: no.
- Abnormal responses: none.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The 24h EC50: 0.87 mg/L, (95 % confidence limits: 0.70 – 0.99 mg/L)
- Other: The date of the last study (Study Code: 20/042-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 05 - 06 March 2020 (tested at least twice a year to demonstrate satisfactory test conditions).

Reported statistics and error estimates:
The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by SPSS PC+ software. The 48 hours EC100 values of the test item were determined directly from the raw data.
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.

Table 3. summary of results

Parameter

24 h EC50

48 h EC50

48h EC100

48h NOEC

48h LOEC

% saturated solution of the 100 mg/L nominal loading rate

1.57

0.82

2.5

0.4

1.0

95 % conf. limits

1.18 - 2.25

0.65 – 0.98

-

-

-

 

Table 4. Number and percentage of immobilised animals


Nominal concentration

[% saturated solution]

Number of treated animals

Immobilised animals

24 hours

48 hours

number

percent

number

percent

Control

20

0

0

0

0

0.064

20

0

0

0

0

0.16

20

0

0

0

0

0.4

20

0

0

0

0

1.0

20

7*

35

15*

75

2.5

20

14*

70

20*

100

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

 

Table 5.Immobilisation of the test animals

Nominal concentration
[% saturated solution]

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

0.064

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

0.16

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

0.4

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

1.0

1

5

4

5

2

5

1

2

3

5

1

4

4

5

1

4

2.5

1

5

4

5

2

5

2

5

3

5

4

5

4

5

4

5

 

 

Table 6. Test conditions during the Test

Parameter

Exposure Time

0 h
(fresh media)

24 h
(old media)

24 h
(fresh media)

48 h
(old media)

temperature (ºC)

20.8

20.4

20.7

20.5

Oxygen concentration (mg/L)

Control

8.6

9.0

8.7

8.8

0.064

8.5

9.0

8.4

8.8

0.16

8.6

8.9

8.5

8.8

0.4

8.6

8.9

8.5

8.7

1.0

8.5

8.8

8.5

8.9

2.5

8.5

8.9

8.5

8.7

pH

Control

7.88

7.89

7.93

7.82

0.064

7.91

7.89

7.95

7.82

0.16

7.91

7.92

7.95

7.87

0.4

7.91

7.90

7.94

7.87

1.0

7.91

7.92

7.95

7.92

2.5

7.91

7.93

7.95

7.94

Validity criteria fulfilled:
yes
Remarks:
There was no immobilization 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 48h-LL50 of the test item in zebrafish was found to be 0.82 mg/L and the NOELR 0.4 mg/L (nominal concentrations).
Executive summary:

The acute toxicity of the test item on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system, following OECD 202 under GLP conditions. Because significant toxic effect was observed at the highest concentration levels (1.0, 10.0 and 100.0 % saturated solutions) during the preliminary range-finding test (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) five test concentrations in a geometric series with a separation factor of 2.5 and one untreated control were tested in the main experiment under semi-static conditions. The nominal concentrations of test item used in the main experiment were: 0.064, 0.16, 0.4, 1.0 and 2.5 % saturated solutions. According to the given expert statement, the biological results are based on the nominal test item concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each, were used for all the test concentrations and the control. The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis, and the 48h EC100 values of the test item were determined directly from the raw data. 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. All validity criteria were met. Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of DCC were as follows: The 48h-LL50 of the test item in zebrafish was found to be 0.82 mg/L (95% CL: 0.65 - 0.98 mg/L) and the NOELR 0.4 mg/L (nominal concentrations, based on mobility).

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE : EPI-SUITE EPA (USA)

2. MODEL: ECOSAR v1.11

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS Number: 538-75-0
SMILES: N(=C=NC(CCCC1)C1)C(CCCC2)C2

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: The QMRF is available in "Attached justification".

5. APPLICABILITY DOMAIN : The QPRF is available in 'Attached justification'.

6. ADEQUACY OF THE RESULT : The QPRF is available in 'Attached justification'.
Guideline:
other: REACH Guidance on QSARs R.6
GLP compliance:
no
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.007 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: calculated value.

ECOSAR v1.11 predicted that the substance has a 48h-LC50 = 0.007 mg/L in daphnia.

Validity criteria fulfilled:
not applicable
Conclusions:
The calculated 48h-LC50 of the test item in daphnia is 0.007 mg/L.
Executive summary:

ECOSAR v1.11 calculation of the 48h-LC50 in daphnia was used. According to this method, the 96h-LC50 = 0.007 mg/L of test item in daphnia.

Description of key information

Key study: Method according to OECD 202, GLP study. The 48h-EL50 of the test item in zebrafish was found to be 0.82 mg/L (95% CL: 0.65 - 0.98 mg/L) and the NOELR 0.4 mg/L (nominal concentrations, based on mobility).

Supporting study: ECOSAR v1.11, accepted calculation method. The substance has a predicted 48h-LC50 = 0.007 mg/L in daphnia.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.007 mg/L

Additional information