Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate

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:

03 June 2016 - 26 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of certificate: 3 Nov 2015

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellow/brown powder
- Storage conditions: in refrigerator (2-8°C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=48
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤ -15 °C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: All test concentrations were prepared separately at loading rates of 1.0, 10 and 100 mg/L by applying three days of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters (Whatman;RC55) where after the Water Accommodated Fractions (WAFs) were used for testing. The final test solutions were clear and increasingly yellow with increasing loading rate.
- Controls: test medium without test item or additives

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by acyclical parthenogenesis under specified breeding conditions. 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 and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started 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. After 7 days of cultivation half of the medium was renewed twice a week.
- Medium other than test medium: yes, M7
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 CaCO3 mg/L

Test temperature:

20-21°C

pH:

8.0 - 8.5

Dissolved oxygen:

8.8 - 9.1 mg O2/L

Nominal and measured concentrations:

Nominal test concentrations: 1.0, 10 and 100 mg/L
Measured test concentrations: 100 mg/L at t=0: 6.48 mg/L; 100mg/L at t=48: 6.32 mg/L
Given the stability of test item in test medium (98% after 48 hours), results were based on the initial measured exposure concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, open, fill volume: 80 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels for the control and the 100 mg/L WAF: 4
- No. of vessels for the 1.0 and the 10 mg/L WAFs: 2

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Culture medium different from test medium: yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, for the 100 mg/L WAF and the control. Temperature of the medium: continuously in a temperature control vessel beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (April 2016)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 6.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- The final result was based on the intitial measured exposure concentration
- No biological, behavioural or other abnormalities were observed.
- Effect concentrations exceeding solubility of substance in test medium: yes

- Recovery: measured concentration at t=48 was 98% of initial measured concentration (loading rate of 100 mg/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
- 48h-EC50: 0.29 mg/L (95%-CI: 0.26 - 0.31 mg/L)
- Other: responses were below the ranges of expected responses at the differen concentrations. Hense, the sensitivity of this batch of Daphnia magna was slightly above the sensitivity of batches previously tested. However, because no effects were seen at the highest tested concentration this higher sensitivity does not affect the results of the concerned study.

Any other information on results incl. tables

Table 1 Number of introduced daphnids and incidence of immobility

Time	Replicate	WAFs prepared at the given loading rate (mg/L)
		Control	1.0 (n.m.)	10 (n.m.)	100 (6.5)
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0

( ) Initial concentration

n.m. Not measured

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the control, no daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels (i.e. 8.8 and 8.9 mg/L).

Conclusions:

The 48h-EC50 was >6.5 mg/L, which was considered to be the maximum solubility limit of Disodium 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonate in test medium.

Executive summary:

A study was performed to assess the effect of Disodium 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonate on the mobility of Daphnia magna after 48 hours. The study was conducted in accordance with OECD 202 and GLP, under static conditions.

 All test concentrations were prepared seperately at loading rates of 1.0, 10 and 100 mg/L. After three days of stirring the aqueous mixtures were filtered through 0.45 µm membrane filters where after the Water Accommodated Fractions (WAFs) were used for testing.

 In a combined limit/range-finding test 20 Daphnia magna (less than 24 hours old) were exposed to a control group and a WAF prepared at 100 mg/L for 48 hours in all-glass, open vessels. Additionally, 10 Daphnia magna were exposed to WAFs prepared at 1.0 and 10 mg/L, under the same experimental conditions. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken at t=0 and at t=48 from the 100 mg/L WAF were analysed. Measured concentrations were 6.5 mg/L at t=0 and 6.3 mg/L at t=48. These results showed that test item remained stable during the 48 -hour test period (98% of the initial concentration after 48 hours).

No immobility was recorded at any of the measured concentrations or the control during the 48 -hour test period. The final result was based on the intitial exposure concentration. The 48h-EC50 was >6.5 mg/L, which was considered to be the maximum solubility of test item in test medium. All acceptability criteria were fulfilled and the study was considered to be valid.