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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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 203 and EU Method C1 and in accordance with the Principles of Good Laboratory Practices (GLP), stability of the tested material was around the detection limit around 24 hours

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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:

yes

Details on sampling:

Stability of the exposure concentration under test conditions was determined by analyzing samples taken from the WAF prepared at 1000 mg/l during the range-finding study.
Sampling:
Frequency: at t=0 hour, t=24 hours and t=96 hours
Volume: 10 ml from the approximate centre of the test vessel.
Storage: samples were transferred to analytical chemistry on the day of sampling
During the final test duplicate samples were taken from the WAF's prepared at 0, 10, 32 and 100 mg/l.
Sampling:
Frequency: at the start of the exposure (t=0 hour)
Volume: 10 ml from the approximate centre of the test vessel.
Storage: samples were transferred to analytical chemistry on the day of sampling
Additionally, reserve samples of 25 ml were taken from all test solutions. These samples were stored at -20 °C for possible analyses

Vehicle:

yes

Details on test solutions:

The procedure for preparation of the exposure solutions was as follows: weighed amounts (29.2, 52.1, 100.6, 166.9 and 311.7 mg) of test substance were spread out on microscopic slides and these amounts corresponded to the nominal concentrations to be tested. Each slide was put in 3 liters of ISO medium and the test material melted in a water bath at a temperature of ca. 60°C.
Each slide with the test substance was put in the required volume of ISO-medium, which was subsequently stirred for ca. 66 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):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Strain: carpio
- Source: Zodiac profacc, "De Haar Vissen", L.U. Wageningen, the Netherlands
- Length at study initiation (length definition, mean, range and SD): 2.0 ± 0.16 cm
- Weight at study initiation (mean and range, SD): 0.24 ± 0.05 g
- Feeding during test: no feeding from 24 hours prior to the tes and during the total test period

ACCLIMATION
- Acclimation period: at least 14 days after delivery
- Acclimation conditions (same as test or not): yes
- Type and amount of food: daily with Trouvit or Artemia

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

not applicable

Hardness:

no information available

Test temperature:

22 - 23 °C

pH:

7.4 - 8.1

Dissolved oxygen:

> 7 mg/l for all concentrations

Salinity:

not applicable

Nominal and measured concentrations:

Range-finding study -
The intial actual concentrations were 0.014 - 0.017% relative to the nominal concentration of 1000 mg/l. After a period of 96 hours, the concentrations measured in the water accomodated fractions of nominally 1000 mg/l were jus above the detection limit of 41 µg/l.
Main study -
In all the samples taken from 0 mg/l, the amount of test substance was below the detaction limit limit of 10 µg/l. At the start of the study, the concentration of Mortrace SB Conc. in the water accomodated fraction (WAF) of nominally 10, 32 and 100 µg/l was just below or above the detection limit of 10 µg/l. Therefore, samples at 96 hour were considered to be of no relevance.

Details on test conditions:

TEST SYSTEM
- Test vessel: 8 litre glass vessel
- Aeration: continuus aeration during the test
- No. of organisms per vessel: 10 fish per concentration
- Biomass loading rate: 0.8 g fish/litre, i.e. 10 fish per 3 litres of test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod

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:

pentachorophenol

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 10 µg/L

Nominal / measured:

not specified

Conc. based on:

other: water accomodated fraction equivalent to the minimum detection limit of Mortrace SB Conc. in water

Basis for effect:

mortality (fish)

Remarks on result:

other: not calculable

Details on results:

In the range finding study, all fish exposed to the WAF's prepared at 100 and 1000 mg/l died within 24 hours and at 10 mg/l, no mortality was noted.
In the main study, after 96 hours of exposure, 70% mortality was recorded in the WAF prepared at 100 mg/l, whereas 90% mortality was found in the WAF prepared at 56 mg/l. In the WAF prepared at 32 mg/l, a total of 30% mortality was found and no mortality was noted in the WAF prepared at 10 and 18 mg/l.
Effects other than mortality were noted in the fish exposed to WAF's prepared at and above 32 mg/l and these were discolouring, hypoactivity, swimming at the bottom, loss of equilibrium and immobility. No effects were noted at the lower WAF's prepared at 10 and 18 mg/l.

Results with reference substance (positive control):

The 96-hour LC50 was in between 0.18 and 0.32 mg/l.

Reported statistics and error estimates:

standard statistical methods were employed

Sublethal observations / clinical signs:

None

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 96-hour LC50 for carp exposed to Mortrace SB Conc. approximated the minimum detection level of Mortrace SB Conc. in water (10 µg/l).

Executive summary:

In the range finding study, all fish exposed to the WAF's prepared at 100 and 1000 mg/l died within 24 hours and at 10 mg/l, no mortality was noted.

In the main study, after 96 hours of exposure, 70% mortality was recorded in the WAF prepared at 100 mg/l, whereas 90% mortality was found in the WAF prepared at 56 mg/l. In the WAF prepared at 32 mg/l, a total of 30% mortality was found and no mortality was noted in the WAF prepared at 10 and 18 mg/l.

Effects other than mortality were noted in the fish exposed to WAF's prepared at and above 32 mg/l and these were discolouring, hypoactivity, swimming at the bottom, loss of equilibrium and immobility. No effects were noted at the lower WAF's prepared at 10 and 18 mg/l.

Under the conditions of the study, the 96-hour LC50 for carp exposed to Mortrace SB Conc. approximated the minimum detection level of Mortrace SB Conc. in water (10 µg/l).

Description of key information

The 96-hour LC50 value of 10 μg/L for Mortrace SB Conc. is based on measured concentrations of the test material (near the detection limit) in a static exposure study with carp (Cyprinus carpio). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

10 µg/L

Additional information

Two valid acute freshwater fish studies are available for Mortrace SB Conc. The key study was assigned a Klimisch score of 2 based on the uncertainty with the mean measured concentrations of Mortrace SB Conc., which were near the analytical method’s detection limit of 10 μg/L. The study was conducted with water accommodated fractions of the test material. The test species was the carp (Cyprinus carpio),and the reported 96-hour LC50 values were circa 10 μg/L. In another supporting, acute toxicity study with zebrafish (Danio rerio) (Klimisch score =2), water accommodated fractions of Mortrace SB Conc. were prepared and juvenile zebrafish were exposed for 96 hours under static renewal conditions. Under this scenario, no mortality was observed in the water accommodated fraction prepared at 1000 mg/L. The mean measured concentrations of Mortrace SB Conc. in the water accommodated fraction in this study was 0.0625 μg/L, the limit of quantification of the HPLC analytical method.