4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

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:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study methodology followed was as per OECD TG 202 and EU Method C2 and in accordance with the Principles of Good Laboratory Practices (GLP), analytical analyses of the tested concentrations was not performed.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

- Range finding test concentrations: WAF's prepared at 10, 100 and 1000 mg/l
- Main study test concentrations: WAF's prepared at 10, 18, 32, 56 and 100 mg/l

Vehicle:

yes

Details on test solutions:

The procedure for preparation of the exposure solutions was as follows: weighed amounts (19.2, 36.7, 64.7, 55.6 and 97.9 mg) of test substance were spread out on microscopic slides and these amounts corresponded to the nominal concentrations to be tested. These microscopic slides with thetest material were melted in a water bath at a temperature of ca. 60°C. (the first 3 slides with the test substance were put in 2 liters of ISO-medium, the last two in 1-litre of ISO medium).
Each slide with the test substance was put in the required volume of ISO-medium, which was subsequently stirred for ca. 91 hours, untl the start of the test. After stirring, the microscopic slides were removed and the solutions were filtered through a paper filter to remove little drops of the test substance that had come off from the slides. The final test solutions were clear without precipitation and the intensity of the resulting water accomodated fractions (WAF's) increased with concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: magna
- Source: in-house
- Age at study initiation: < 24 hours
- Method of breeding: With new born daphnids, i.e. < 3 days old by placing about 250 of these into 10 litres of medium in glass culture vessels.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: after 7 days of cultivation, half of the medium twice a week
- Feeding during test: no
- Food type during breeding: daily, a suspension of fresh water algae
- Temperature of medium: 18-22 °C, constant within ± 1 °C

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

not applicable

Hardness:

no information

Test temperature:

20 - 23 °C

pH:

ranged between 8.0 - 8.3

Dissolved oxygen:

ranged between 8.7 - 10.0 mg/l

Salinity:

not applicable

Nominal and measured concentrations:

Sampling for analysis of actual expoure concentrations at the end of the test was cancelled as samples taken during the fish toxicity test revealed only detectable amounts of test substance in the WAF's of 1000 mg/l (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml glass vessel
- Aeration: no aeration
- No. of organisms per vessel: 20/test concentration
- Loading: 10 per vessel containing 100 ml medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium (as per standard recommendations)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED: Immobilization was noted

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: WAF's prepared at 10, 100 and 1000 mg/l
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

32 - 56 mg/L

Nominal / measured:

not specified

Conc. based on:

other: water accomodated fraction

Basis for effect:

mobility

Remarks on result:

other: 95% fiducial limits could not be calculated using the Finney model as the index of regression significance was > 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

not specified

Conc. based on:

other: water accomodated fraction

Basis for effect:

mobility

Remarks on result:

other: The 48-hour EC50 corresponded with a WAF prepared at 45 mg/l with 95% fiducial limits of WAF's prepared at 42.3 mg/l and 49.8 mg/l.

Details on results:

Range finding test- Immobility was observed in all the daphnids exposed to the WAF's prepared at 100 and 1000 mg/l. No immobility was seen in the WAF prepared at 10 mg/l.

Main study - After 24 hours of exposure the total rate of immobilization was 55% in both the WAF's prepared at 56 and 100 mg/l. No immobilization of the daphnids was noted in the WAF's prepared at 32 mg/l or lower concentrations. Some of the daphnids present in the WAF's prepared at WAF's prepared at 32 mg/l or higher were found trapped at the surface and were re-immersed.

After 48 hours of exposure immobilization of all the daphnids were noted in the WAF prepared at 100 mg/l. The total incidence of immobility among the daphids exposed to the WAF prepared at 56 mg/l had increased to 90% and no significant (>10%) immobilization was seen at WAF's prepared at 32 mg/l or lower.

The pH varied between 8.0 - 8.3 and oxygen concentrations were found to be > 8 mg/l for all measurements and the temperature varied between 20 - 23 °C. In the controls, no daphnia became immobilized or trapped at the surface of the water.
The 24-hour EC50 corresponded with a WAF prepared between 32 and 56 mg/l and 95% fiducial limits could not be calculated using the Finney model as the index of regression significance was > 1. The 48-hour EC50 corresponded with a WAF prepared at 45 mg/l with 95% fiducial limits of WAF's prepared at 42.3 mg/l and 49.8 mg/l. The NOEC with respect to mobility of daphnia was found to correspond with a WAF prepared at a concentration of 32 mg/l.

Results with reference substance (positive control):

The 24-hour EC50 was 0.57 mg/l (95% fiducial limits: 0.48 - 0.73 mg/l) and the 48-hour EC50 was 0.40 mg/l (95% fiducial limits: 0.35 - 0.49 mg/l).

Reported statistics and error estimates:

standard statistical methods were employed

None

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study Mortarce SB Conc. did not induce acute immobilization of daphnia magna exposed to WAF prepared at a concentration of 32 mg/l or lower. The 48-hour EC50 corresponded with a WAF prepared at 45 mg/l with 95% fiducial limits of WAF's prepared at 42.3 mg/l and 49.8 mg/l. The NOEC with respect to mobility of daphnia was found to correspond with a WAF prepared at a concentration of 32 mg/l.

Executive summary:

In this study, daphnids were exposed for a maximum of 48 hours to a range of water accomodated fractions (WAF's ) prepared at 10, 18, 32, 56 and 100 mg/l. Weighed amounts of the test substance were spread out on microscopic slides and placed in the test media, which were stirred for ca. 91 hours until the start of the exposure. After stirring, the slides were removed and the solutions were filtered through a filter paper. The final test solutions were all clear and light brown with the intensity of the colour increasing with concentration. The test was perfomed in duplicate with 10 daphnids per vessel.

Under the conditions of the study Mortarce SB Conc. did not induce acute immobilization of daphnia magna exposed to WAF prepared at a concentration of 32 mg/l or lower. The 48-hour EC50 corresponded with a WAF prepared at 45 mg/l with 95% fiducial limits of WAF's prepared at 42.3 mg/l and 49.8 mg/l. The NOEC with respect to mobility of daphnia was found to correspond with a WAF prepared at a concentration of 32 mg/l.

Description of key information

In an acute toxicity study with Daphnia magna, the 48-hour EC50 value for Mortrace SB Conc. corresponded with a water accommodated fraction of 45 mg/L. Actual measurement of  Mortrace SB Conc. were not undertaken in this study. However, the concentrations of Mortrace SB Conc. in the water fraction corresponding to the 48-hour EC50 are expected to be similar to those determined in the fish acute toxicity study (i.e. 10 μg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 µg/L

Additional information

One static acute toxicity test with Daphnia magna was available for Mortrace SB Conc. This study was assigned a Klimisch score of 2 based on the lack of analytical measures of test solution concentrations. The lack of measured concentrations in this study was based on minimal detection in the fish acute toxicity test with Mortrace SB Conc. The 48-hour EC50 value (based on immobility) was reported as 45 mg Mortrace SB Conc/L. This value was based on initial nominal loading rate of the water accommodated fraction, however the measured water concentration of Mortrace SB Conc.at this loading rate are expected to be much lower, likely in the range of 10 μg/L as was seen in the fish acute toxicity study. Thus, the key parameter for the acute invertebrate endpoint was the EC50 value of 45 mg/L, which, based on the acute toxicity test with the fish, was assumed to be approximately 10 μg/L in the water column.