Hydrogen [4-[4-(p-ethoxyanilino)-4'-[ethyl(m-sulphonatobenzyl)amino]-2'-methylbenzhydrylene]-3-methylcyclohexa-2,5-dien-1-ylidene](ethyl)(m-sulphonatobenzyl)ammonium, monosodium salt

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-09-15 to 2014-11-13

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

Principles of method if other than guideline:

Guideline followed

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.

Analyses: The concentrations of the test item were analysed in the duplicate test media samples from the filtrate and the two highest dilutions of bothsampling times (0 and 48 hours). From the control samples both of the duplicate samples was analysed from each of both sampling times.

Vehicle:

no

Details on test solutions:

Test Concentrations: Filtrate of 100 mg test item/L, 1:2, 1:4, 1:8 and 1:16 dilutions, corresponding to 3.82, 1.65 and 0.829 mg test item/L for the three highest test concentrations and a control.
Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: A stock solution was prepared by dissolving 99.9 mg test item into 999 mL test water. The stock suspension was stirred for 23.5 hours in the dark to dissolve as much test item as possible. Then, the undissolved test item was separated by filtration (0.45 µm cellulose nitrate filter) and the required test concentrations were prepared by diluting adequate volumes of this test medium with test water. The test media were prepared just before introduction of the daphnids (= start of the test).

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 3.75 to 19.5 hours old
Sex: Female
Origin: The daphnids introduced in the test were taken from IBACON's in house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of IBACON under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in the same reconstituted water of a similar quality with regards to pH, the constituent salts and total hardness as the test water used in the test. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of IBACON.
Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

20 to 21 °C at test start;
20 to 21 °C at test end

pH:

7.7 to 7.9 at test start;
7.6 to 7.7 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

7.2 to 9.2 mg/L at test start;
7.4 to 7.9 mg/L at test end

Nominal and measured concentrations:

Filtrate of 100 mg test item/L, 1:2, 1:4, 1:8 and 1:16 dilutions, corresponding to 3.82, 1.65 and 0.829 mg test item/L for the three highest test concentrations and a control.

Details on test conditions:

Test Environment: Controlled environment room

Measurement of pH, Dissolved Oxygen and Water Temperature: The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
- Water Temperature: 20 to 21 °C at test start; 20 to 21 °C at test end
- pH-Values: 7.7 to 7.9 at test start; 7.6 to 7.7 at test end; and thus the pH-value did not vary by more than 1.5 units
- Dissolved Oxygen Concentration: 7.2 to 9.2 mg/L at test start; 7.4 to 7.9 mg/L at test end
- Light Regime: 16 h light : 8 h dark
- Light Intensity: The light intensity was 820 to 1060 lux (measured once during the test).

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.48 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

0.993 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.808 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.829 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 0.829 mg test item/L. At the concentration of 1.65 mg test item/L, 17 animals were immobile and 18 animals were immobile at the concentration of 3.82 mg test item/L.

Results with reference substance (positive control):

Results of the most recent Test with the Reference Item Potassium dichromate performed in July 2014 (88292220)
Nominal Concentration % of immobilised daphnids after
[mg test item/L] 24 hours 48 hours
Control 0 0
0.125 0 0
0.25 0 0
0.5 0 0
1.0 25 70
2.0 95 100
EC50 [mg/L]: 1.225 0.946
95 % CI [mg/L]: 1.043 - 1.451 n.d.
EC20 [mg/L]: 0.956 0.865
95 % CI [mg/L]: 0.734 - 1.113 n.d.
EC10 [mg/L]: 0.840 0.825
95 % CI [mg/L]: 0.594 - 0.995 n.d.
NOEC [mg/L]: 0.5 0.5
LOEC [mg/L]: 1.0 1.0
Values refer to nominal test concentrations; CI: Confidence interval; n.d.: not determinable; NOEC and LOEC were determined directly from the raw data.

In the most recent test with the reference item potassium dichromate performed in July 2014 (study code 88292220) the EC50 after 24 hours was determined to be 1.225 mg test item/L indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50- 24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
For the statistic, the two lowest concentrations were extrapolated with the recovery determined for the lowest measured concentration.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat(R) Solutions GmbH.

Influence of FAT 20085/B TE on the Mobility of Daphnia magna
Endpoint [mg test item/L]	24 hours	48 hours
EC50	2.48	1.48
95 % CI	1.91 - 3.55	n.d.
EC20	1.37	0.993
95 % CI	0.917 -1.78	n.d.
EC10	1.00	0.808
95 % CI	0.573 -1.35	n.d.
NOEC	0.829	0.829
LOEC	1.65	1.65
Values refer to mean measured test concentrations CI: Confidence interval n.d.: not determinable NOEC and LOEC were determined directly from the raw data.

Analytical Results

sample description	concentration	RSD
[mg/L]	found [mg/L]	[%]	n
control	n.a.	n.a.	4
dilution 1:4	0.829	12	4
dilution 1:2	1.65	10	4
Filtrate of 100 mg/L	3.82	10	4

RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

Validity criteria fulfilled:

yes

Conclusions:

The 48h EC50 value was calculated to be 1.48 mg/L.

Executive summary:

The acute toxicity of the test item to Daphnia magna in a static 48-hour immobilisation test was determined and based on the procedures indicated by the following internationally accepted methods:

- OECD Guideline No 202

- EU Method C.2

This study encompassed 6 treatment groups (5 dose rates of the test item, control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation

after 24 and 48 hours.Filtrate of 100 mg test item/L, 1:2, 1:4, 1:8 and 1:16 dilutions, corresponding to 3.82, 1.65 and 0.829 mg test item/L were the three highest test concentrations.

Biological Results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 0.829 mg test item/L. At the concentration of 1.65 mg test item/L, 17 animals were immobile and 18 animals were immobile at the concentration of 3.82 mg test item/L.

Analytical Results:

At the start of the test 4.126, 1.779 and 0.904 mg test item/L were found in the filtrate and in the 1:2 and 1:4 dilution (average concentrations, the other dilutions were not analysed since they were below the NOEC). After 48 hours test duration, 3.513, 1.522 and 0.753 mg test item/L were found in the filtrate and in the 1:2 and 1:4 dilution (average concentrations, the other dilutions were not analysed since they were below the NOEC).

Therefore, all reported results refer to measured concentrations.

Conclusion:

The toxic effect of the test item FAT 20085/B TE to Daphnia magna was assessed in a static dose-response test. The 48-hour NOEC was determined to be 0.829 mg test item/L. The 48-hour LOEC was determined to be 1.65 mg test item/L and the 48-hour EC₅₀ value was calculated to be 1.48 mg test item/L.

This study is classified acceptable and satisfies the guideline requirements for daphnia acute test studies.

Description of key information

The 48h EC50 of FAT 20085/B TE to Daphnia magna in a static immobilisation test was found to be 1.48 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.48 mg/L

Additional information

The acute toxicity of the test item to Daphnia magna in a static 48-hour immobilisation test was determined and based on the procedures indicated by the following internationally accepted methods:

- OECD Guideline No 202

- EU Method C.2

This study encompassed 6 treatment groups (5 dose rates of the test item, control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation

after 24 and 48 hours.Filtrate of 100 mg test item/L, 1:2, 1:4, 1:8 and 1:16 dilutions, corresponding to 3.82, 1.65 and 0.829 mg test item/L were the three highest test concentrations.

Biological Results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 0.829 mg test item/L. At the concentration of 1.65 mg test item/L, 17 animals were immobile and 18 animals were immobile at the concentration of 3.82 mg test item/L.

Analytical Results:

At the start of the test 4.126, 1.779 and 0.904 mg test item/L were found in the filtrate and in the 1:2 and 1:4 dilution (average concentrations, the other dilutions were not analysed since they were below the NOEC). After 48 hours test duration, 3.513, 1.522 and 0.753 mg test item/L were found in the filtrate and in the 1:2 and 1:4 dilution (average concentrations, the other dilutions were not analysed since they were below the NOEC).

Therefore, all reported results refer to measured concentrations.

Conclusion:

The toxic effect of the test item FAT 20085/B TE to Daphnia magna was assessed in a static dose-response test. The 48-hour NOEC was determined to be 0.829 mg test item/L. The 48-hour LOEC was determined to be 1.65 mg test item/L and the 48-hour EC₅₀ value was calculated to be 1.48 mg test item/L.

This study is classified acceptable and satisfies the guideline requirements for daphnia acute test studies.