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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June - 09 July 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results. However, since the procedural recovery at 0.1 mg/L is poor (36% whereas it should be 70 - 110%), the study is considered to be reliable with restrictions (KC2). It has still been demonstrated that the concentrations to which the Daphnia were exposed has was higher than the max. solubility of the substance and therefore the conclusion that there are no effects below the max. solubility in test medium remains valid.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
(2004)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): IN-1000
- Appearance: Dark amber liquid
- Storage condition of test material: In refrigerator (2-8°C) container flushed with nitrogen





Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Test concentrations were verified by chemical analysis. Water samples (2 mL) were taken from the approximate centre of the test vessels of the control and each exposure level at t = 0 and t = 48 hours.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Samples were stored in the freezer until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
All test solutions were prepared separately and started with loading rates ranging from 1.0 to 100 mg/L. Two days of magnetic stirring was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were siphoned through glass wool where after the resulting Water Accommodated Fractions (WAFs) were used for testing. The final test solutions were all clear and colourless except for the WAF prepared at a loading rate of 100 mg/L for the combined limit/range-finding test that was observed to be slightly hazy.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus 1820)
- Source: in-house laboratory culture with a known history (at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions)
- Age at study initiation: young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old
- Method of breeding: start of each 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. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week.
- Feeding during test: no


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg/L (CaCO3)
Test temperature:
20 - 21°C
pH:
7.8-8.2
Dissolved oxygen:
8.6 - 8.9 mg O2/L
test end: max. 8.9 mg O2/L
Nominal and measured concentrations:
Based on the results of the range-finding test (0, 10 and 75% immobility observed in WAFs prepared at 1.0, 10 and 100 mg/L, respectively), WAFs with the following loading rates were prepared in the final test: 4.6, 10, 22, 46 and 100 mg/L.
Measured test concentrations: see tables 1 and 2 in field 'Any other information on results'
Details on test conditions:
TEST SYSTEM
The tests were performed in all-glass vessels (100 mL) containing 80 mL of test solution.

* Combined limit/ range-finding test (static):
- Three test concentrations (WAFs prepared at a loading rate of 1.0, 10 and 100 mg/L) plus one control
- control and WAF at 100 mg/L: each in 4 replicates, 5 daphnids per replicate (i.e. 20 animals/concentration)
- WAF at 1.0 and 10 mg/L: each in duplicate with 5 animals/test vessel (i.e. 10 animals/concentration).

* Final test (static):
- Six test concentrations (WAFs prepared at a loading rate of 4.6, 10, 22, 46 and 100 mg/L) plus one control each in 4-fold with 5 animals/test vessel (i.e. 20 animals/concentration).
- Loading rate: 16 mL test solution per organism
- Test vessels were maintained at 20 °C under a photoperiod of 16 hours light : 8 hours dark and without supplementary aeration or feeding during the 48 hour exposure period.
- The temperature was measured continuously and the pH and dissolved oxygen levels were recorded at the start and at the end of the study for all concentrations and the control.

TEST MEDIUM / WATER PARAMETERS
- Test medium: adjusted ISO
- Culture medium different from test medium: yes (M7)

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: Immobility (at t=24 and t=48 hours): Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (29 June - 01 July 2015)

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Basis for effect:
mobility
Remarks on result:
other: The EC50 was > maximum soluble concentration of test substance in medium (440 µg/L)
Details on results:
* Range-finding test:
At t = 0 the measured concentrations were 0.85 and 16. 1 mg/L and at t= 48h the measured concentrations were 0.186 (22% of initial) and 0.806 mg/L(5.0% of initial) in WAFs prepared at 10 and 100 mg/L, respectively.

* Final test:
Measured concentrations ranged from 10 - 15 % of inital at the end of the test. See Table 1.

The geometric mean measured concentration at the highest loading rate was calculated to correspond with 440 µg/L.

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 48-h EC50: 0.36 mg/L

Any other information on results incl. tables

Table 1: Concentrations of the test substance in test medium - first final test

Time of sampling
[hours]

Loading rate1

[mg/L]

Concentration
analysed
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

 

46

1.20

 

 

100

1.16

 

 

 

 

 

48

0

n.d.

 

 

46

0.122

10

 

100

0.170

15

 

 

 

 

1          A water accommodated fraction (WAF) prepared at the loading rate.

n.d.    Not detected.

Table 2:          Geometric mean measured concentration versus WAFs prepared at various loading rates

IN-1000, WAF prepared at the given loading rate (mg/L)

Measured concentrations (mg/L)

Geometric mean measured concentration (mg/L)

t=0h

t=48h

46

1.20

0.122

0.38

100

1.16

0.170

0.44

Range-finding test

After 48 hours of exposure 75% of the daphnids exposed to the WAF prepared at a loading rate of 100 mg/L were immobilized. No (significant) immobility was observed in the WAFs prepared at loading rates of 1.0 and 10 mg/L and in the control.

 

Final test

Table 3 shows the responses recorded during the final test. After 48 hours of exposure 40% of the daphnids exposed to the WAF prepared at a loading rate of 100 mg/L were immobilized. This was not in agreement with the results of the combined/limit range-finding test where 75% of the daphnids became immobilized. However, taking the measured concentrations into account, i.e. 16 mg/L initially in the combined limit/range-finding test and 1.2 mg/L initially in the final test, the observed effects are plausible.

 

Based on the observed haziness of the WAF prepared at a loading rate of 100 mg/L for the combined limit/range-finding test it was expected that the test concentration was above the solubility limit in test medium. The fact that test concentrations in the WAFs prepared at loading rates of 46 and 100 mg/L for the final test were similar indicated that the maximum solubility in test medium was approximately 0.4 mg/L.

Table 3: Cumulative immobilisation data for Daphnia magna exposed for 48 hours to the test substance

Time (h)

Replicate

IN-1000, WAF prepared at the given loading rate (mg/L)

Control

4.6

10

22

46

100

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 [2]

0

0

1

B

0

0

0

0

0

0

C

0

0

0

0

0

1

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

2

Effect %

0

0

0

0

0

10

 

 

 

 

 

 

48

A

0

0

0

0

0

3

B

0

0

0

0

0

1

C

0

0

0

0

0

2

D

0

0

0

0

0

2

Total immobilised

0

0

0

0

0

8

Effect %

0

0

0

0

0

40

[ ]: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

 A, B, C, D = replicate 1, 2, 3, 4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
<10% immobilization in control group, O2 conc. at end is >=3 mg/L in control and test vessels
Conclusions:
The 48h-EC50 exceeded a WAF prepared at a loading rate of 100 mg/L that corresponded with a geometric mean measured concentration of 440 µg/L. It can consequently be stated that IN-1000 is not acutely toxic up to and including its limit of solubility in test medium under the conditions of the present static test.
Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna under static conditions. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP.

Groups of twenty daphnids (less than 24 hours old) were exposed for 48 hours to five WAFs of the substance dispersed in test water (loading rates 4.6 - 100 mg/L) and a control. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. The measured concentration at t = 48h was 10% of initial at a loading rate of 46 mg/L and 15% of initial at a loading rate of 100 mg/L. The geometric mean measured concentration at the highest loading rate was calculated to correspond with 440 µg/L. The 48h-EC50 exceeded a WSF prepared at a loading rate of 100 mg/L. It can consequently be stated that IN-1000 is not acutely toxic up to and including the limit of solubility in test medium under the conditions of the present static test. The study is conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results. However, because of the poor procedural recovery at 0.1 mg/L (36% whereas it should be 70 - 110%) and the fact that the measured concentrations at the end of the test were in this range, this study is considered to be less reliable. Since it has still been sufficiently demonstrated that the organisms have been exposed to concentrations higher than the maximum solubility, KC2 is assigned.