2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

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:

June 19 to June 23, 1995.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP and reported with a certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None specified

Analytical monitoring:

yes

Details on sampling:

Sampling was carried out directly from the test vessels.
Before sampling the test media were mixed with an Ultraturrax (at minimum 3 (min) with 20000 (rpm)).
Sampling was carried out during the mixing procedure to prevent losses of test substance via sedimentation.

Vehicle:

no

Details on test solutions:

The test substance was weighed out and transferred into a measuring flask and filled up to a volume of 1 [L] with dilution water and sonicated for 30 (min) at 40 [°C].
Thereafter it was poured in the test tank. The test medium (volume 20 [L]) was mixed thoroughly, using an Ultraturrax (at minimum 3 [min] with 20000 (rpm)). Then the fish were exposed to the test solution. Mixing of the test medium was repeated every 24 [h].

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST SYSTEM: Zebra Fish (Brachydanio rerio)
Reason for the selection of the test system: According to the guideline Brachydanio rerio is suitable for this kind of study.
Origin: Leine-Zoo, Marktplatz 7 + 11, 0-30880 Laatzen
All fish which were used in the test originated from the same delivery of the supplier.
Holding: Holding was performed in the test facility at 21-25 [°C] and diffuse light (≈ 0.1-10 [μmol/m2s], natural photoperiod daily). The containers were aerated continuously. Water was changed once per week. Zebra Fish with 12 days of acclimatisation were used for the study. During that time mortality was < 5 [%].
Water: Tap water of local origin is used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 [h]. Chemical analysis of the tap water islisted in the study plan.
Feeding: Food was provided 3x per week. The amount of food was 4 [%] of the fish weight. The test organisms were not fed 24 [h] before test begin.
Food: Kronen Fish Allround 2 (mml, company Ssniff Spezialdiåten GmbH

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

Total hardness 136 (mg/L) CaCO3

Test temperature:

Temperature in water ranged from 21.1 to 22.9 [°C] (nominal value: 23 ± 2 [°C])

pH:

pH ranged from 7.86 to 8.05 (nominal value: 7.96 ± 0.9)

Dissolved oxygen:

Oxygen saturation ranged from 86 to 92 [%] (nominal value: 88.7 ± 3.3 [%])

Salinity:

Freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations

Details on test conditions:

TEST SUBSTANCE: Lowinox WSP
Stock solution: No stock solution was prepared.
Test concentration: 100 [mg/L]
Vehicle: None

CONTROL
One control with water (without test substance) was tested under the same test conditions as the test substance. The control was also used in another test.

REFERENCE SUBSTANCE
No reference substance is recommended for this test according to OECD-guideline 203.

TEST METHOD
A static test was performed.
Test duration: 96 [h], June 19 to June 23, 1995

APPLICATION
The test substance was weighed out and transferred into a measuring flask and filled up to a volume of 1 [L] with dilution water and sonicated for 30 [min] at 40 [°C].
Thereafter it was poured in the test tank. The test medium (volume 20 [L]) was mixed thoroughly, using an Ultraturrax (at minimum 3 [min] with 20000 (rpm)). Then the fish were exposed to the test solution. Mixing of the test medium was repeated every 24 ([h].
Frequency and duration of application: 1 x about 96 [h] at the beginning of the test.
Test container: Aquaria with a 15 [L]-volume were used.
Test volume: The test volume was 10 [L] per tank.
Total hardness: 136 [mg/L] CaCO3
Number of fish: 7 Zebra Fish were used per concentration and control; Average length at the test start: 2.27 [cm]; Average weight at the test start: 0.72 [g]
Replicates: One replicate per concentration and control
Stock of fish: Fish density in the tanks was < 1 [g] fish per liter test medium.
Feeding of test organisms: During the test the fish were not fed.
Temperature: Temperature in water ranged from 21.1 to 22.9 [°C] (nominal value: 23 ± 2 [°C])
Light intensity: Light intensity was ≈ 10 [μmol/m2-s]
Photoperiod: A natural photoperiod occurred during the test.

KIND AND FREQUENCY OF MEASUREMENT AND OBSERVATIONS
pH-value, oxygen saturation, and temperature were measured at the beginning of the test and every 24 (h).
The total hardness of the water was determined at the beginning of the test out of the control tank.
Before test start weight and size of a representative number of test organisms which were not used in the test were determined.
Behaviour of fish and mortality were observed daily in all test vessels.
During the test the room temperature was recorded with a thermohygrographe.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

TEST OF IDENTIFICATION AND SOLUBILITY
The identity of the test substance was checked. Agreement was ascertained according to the sponsor with regard to aggregate state, consistency, colour, batch number and name of the test substance.
The solubility of the test substance (1000 [mg/L]) was checked in tap water and with the vehicle Tween 80, (100 [mg/L]). White test substance flocks appeared in all replicates during the whole 96 (h) of the test. As a result of this the study was carried out without vehicle.

RESULTS OF THE INLIFE-PART: MAIN TEST
Mortality and symptoms of toxicity during the test are given in table form (see Any other information). The cumulative mortality at each concentration level and the control at each time of observation is shown in table form (see Any other information).
The test concentration of 100 (mg/L) caused no mortality or lethal effects.
The LC50 is > 100 (mg/L).
Therefore the NOEC was laid down as ≥ 100 (mg/L).

Results with reference substance (positive control):

No reference substance is recommended for this test according to OECD-guideline 203.

Reported statistics and error estimates:

None specified

Sublethal observations / clinical signs:

Preliminary test results: mortality as a function of exposure time

Concentration [mg/L]	Cumulative mortality [%]
	Test duration [h]
	24	48	72	96
100	0	0	0	0
Control	0	0	0	0

 

Observations in the test containers (nominal concentration)

Concentration Effect* [mg/L]	Test duration [h]
	24	48	72	96
100 (1)	7/7	7/7	7/7	7/7
Control (1)	7/7	7/7	7/7	7/7

*) The numbers in brackets corresponded to the following observations: (1) = without results

 

Cumulative mortality in the test container (nominal concentrations)

Concentration [mg/L]	Cumulative mortality [%]
	Test duration [h]
	24	48	72	96
100	0	0	0	0
Control	0	0	0	0

 

Validity criteria fulfilled:

yes

Conclusions:

The test concentration of 100 (mg/L) caused no mortality or lethal effects.
The LC50 is > 100 (mg/L).
Therefore the NOEC was laid down as ≥ 100 (mg/L).

Executive summary:

The acute lethal toxicity to fish (Zebra Fish) was determined in a limit test according to OECD Guideline 203, from June 19 to June 23, 1995.

Test substance was the product Lowinox WSP, Batch No. 5E049. No carrier for dissolving the test substance was used.

The test method was static. The test substance was applied once at the beginning. The test was conducted with the nominal concentration 100 [mg/L] selected on the basis of a preliminary static test. Duration of the test was 96 [h].

7 test organisms were exposed to the test concentration and control.

Turbidity of the test medium was observed throughout the test period.

The test substance concentrations were determined via HPLC. The overall mean value of the recovery rates at the end of the test was 96 (%). Therefore calculations of LC-values were performed based on nominal concentration.

The acute lethal toxicity of the test substance Lowinox WSP, based on the nominal concentration is summarized in the following table:

 

LC values, based on nominal concentrations [mg/L]

Test duration [h]	LC50 [mg/L]
24	>100
48	>100
72	>100
96	>100
LC100 =                                   >100 Lowest concentration of the test substance in the test with 100 [%] mortality after 96 [h]
LC0 =                                          100 Highest concentration of the test substance in the test with 0 [%] mortality after 96 [h]

 

 

On the basis of the observations made during the test, the NOEC was determined at a concentration (nominal) of ≥ 100 [mg/L] of the test substance, Lowinox WSP.

Water quality parameters of temperature, pH, dissolved O2 measured at 0, 24, 48, 72 and 96 [h] were determined to be within the acceptable limits.

The overall ranges were:

daily temperature: 21.1 to 22.9 [°C]

pH: 7.86 to 8.05

O2-saturation: 86 to 92 [%]

Description of key information

Key value determined in laboratory testing to OECD Guideline 203. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

On the basis of the observations made during the test, the NOEC was determined at a concentration (nominal) of ≥ 100 [mg/L] of the test substance, Lowinox WSP.