2-tert-butyl-1,4-dimethoxybenzene

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:

2019-01-28 to 2019-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Specific details on test material used for the study:

Name: 2-tert-butyl-1,4-dimethoxybenzene
Public name: Vetimoss
EC number: 244-216-5
CAS number: 21112-37-8
Batch/Lot Number: A180118C
Description: Clear, pale yellow liquid
Expiry Date: 06 June 2019
Purity: 99.78%
Storage Conditions: Room temperature (15-25°C, ≤70 RH%), under inert gas, protected from humidity (tight closed container)

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

Analytical measurements were performed from the Control and at the applied test concentration levels at the beginning and at the end of each renewal period. Two samples were taken for analytical measurements at 0, 24, 48, 72 and 96 hours into 16-mL screw cap glass vials. One set was analysed (Set 1, ~10 mL) and one as a back-up (Set 2, ~10 mL), the weight of the samples was recorded.

Vehicle:

no

Details on test solutions:

Formulation
Because the Test Item was a UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials), the test solutions were prepared individually using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23 (2000). The saturated Test Item solutions were prepared individually [mg/L Test Item nominal loading rates (WAFs)] by directly dispersing/dissolving the needed amount of Test Item into the test medium (ISO medium) before the start of each renewal period. These solutions were mixed continuously for approximately 1 hour at room temperature. The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solutions without an additional settling time. This procedure was considered to be appropriate following an evaluation of the best practical method for this Test Item. The test solutions were prepared just before introduction of the fish (start of each renewal period).

Untreated Control
The dilution water (circulated and filtered) was used without of addition of the Test Item.

Dose rates
Because mortality was observed during the preliminary range-finding test in the highest applied concentration of 100 mg/L (nominal), five test concentrations in a geometric series (factor 2.0) and one control were used in the Main test. The concentrations in the Definitive Test were based on the results of the Preliminary Range-finding Test and were: 6.25, 12.5, 25, 50 and 100 mg/L nominal loading rates (WAFs).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Brachydanio rerio)
Source: Szent István University, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1. – Hungary
Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test and it is recommended by the test guidelines.
Number of animals: 7 fish in test concentration and control groups
Body length of animals: 2.0–2.5 cm
Acclimatisation: >12 days
Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods; the fish were healthy. Mortality was less than 5% of the population for seven days before the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

144 mg CaCO3/l

Test temperature:

20.1 to 22.5

pH:

7.29 to 8.18

Dissolved oxygen:

82 to 100%

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0, 6.25, 12.5, 25, 50, 100 mg/l
Geometric mean measured concentrations: 0, 1.26, 2.36, 2.85, 3.81, 4.79 mg/l

Details on test conditions:

Water: Circulated and filtered water will be used for holding of fish. During the test the fish will be held in water of the same quality as used for holding.
Feeding: Three times per week or daily under acclimatisation, until 24 hours before the start of the test.
Light: 12 to 16 hours photoperiod daily.

PERFORMANCE
The test duration was 96 hours. One aquarium was used for test groups and control groups, respectively. Each aquarium comprised 7 fish and 5 L test solution. The animals were not fed during the test. The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

Because the Test Item was not stable over the 96h exposure period (based on the results of the analytical method validation: the Definitive Test was carried out in semi-static conditions and the frequency of the renewal periods were 24 hours.

OBSERVATIONS
The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality and sub-lethal effect were determined during the experiment.

The test conditions (pH, temperature, oxygen saturation) were measured at the start and at the end of each renewal period during the test.

The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.26 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

2.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range finding test
In the preliminary test, no mortality was observed at 0, 0.1, 1 and 10 mg/L, with 40% mortality at 100 mg/L.

VALIDITY
The validity criteria were within acceptable limits and therefore the study is considered as valid.

MEASURED TEST ITEM CONCENTRATIONS
The following nominal concentrations were tested: 6.25, 12.5, 25, 50 and 100 mg/L nominal loading rates (WAFs). The LOD was calculated as 0.052 mg/L. The corresponding measured geometric mean Test Item concentrations were: and 1.26, 2.36, 2.85, 3.81, 4.79 mg/L. As the measured Test Item concentration results deviated more than ±20% of the nominal, the biological results are based on these measured concentrations.

SUB-LETHAL EFFECTS
Behaviour of Control fish was found to be normal through the exposure time of 96 hours. Behaviour of fish was found as normal at the lowest concentration level. The most typical clinical signs were localization close to the bottom of the aquarium or close to the surface, dark colour, decreased activity, unbalanced swimming which were observed in all concentrations between 12.5-100 mg/L nominal loading rates (WAFs).

BODY WEIGHT
The body weight of 7 fish was weighed at the start of the test. There was no considerable difference observed concerning body weights between the groups.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The 72 and 96 hours LC50 values were calculatedby Probit analysis using SPSS PC+ software. The 24, 48 hours LC50 value could not be calculated based on the mortality data (it was less than 50%).
 
The 96 hours NOEC, LOEC and LC100were determined directly from the raw data.

Sublethal observations / clinical signs:

Nominal and measured concentrations of the Test Item

Nominal concentrations of the Test Item [1]	Measured concentrationsat the renewal periods (mg/L)								Geometric mean
	1^st		2^nd		3^rd		4^th
	Start	End	Start	End	Start	End	Start	End	(mg/L)
Control	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	---
6.25	1.80	0.82	1.16	0.56	2.13	0.79	3.27	1.20	1.26
12.5	0.99	0.53	5.24	1.91	5.22	1.96	7.70	2.34	2.36
25	5.68	2.09	1.25	0.76	8.58	5.55	---	---	2.85
50	4.40	1.60	4.01	1.91	10.09	5.65	---	---	3.81
100	5.37	2.02	4.94	2.25	12.67	7.88	---	---	4.79

n.d.: not detected

[1]: Nominal concentrations of the Test Item: mg/l loading rates (WAFs)

Cumulative mortality data in the Definitive Test

Nominal concentration (mg/L)	Cumulative mortality [number of fish] (initial population = 7 fish)
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
1.26	0	0	0	0	0	0
2.36	0	0	0	0	0	1
2.85	0	0	0	0	7	7
3.81	0	0	0	0	7	7
4.79	1	1	1	1	7	7

Measured and calculated data of bodyweight

The body weight of 7 fish was weighed at the start of the test. There was no considerable difference observed concerning body weights between the groups.

Nominal concentration (mg/L)	Measured weight of 7 fish(g)	Calculated mean weight of 1 fish(g)	Loading of testing aquarium (g fish/l testing liquid)
Control	1.17	0.17	0.23
1.26	1.32	0.19	0.26
2.36	1.38	0.20	0.28
2.85	1.30	0.19	0.26
3.81	1.39	0.20	0.28
4.79	1.22	0.17	0.24

Conclusion

Under the conditions of this acute fish toxicity studyon Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the Test Item were the following:

Endpoints	Measured concentrations the Test Item	95% confidence limits
24h LC50value [1]	---	---
48h LC50value  [1]	---	---
72h LC50 value	2.59 mg/L	2.40 – 2.80
96h LC50 value	2.51 mg/L	2.33 – 2.76
96h LC100 value	2.85mg/L	---
96h NOEC	1.26	---
96h LOEC	2.36	---

[1] Could not be calculated based onthe mortality data at the 24 and 48 hours observation (it was less than 50%).

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 of Vetimoss in fish is 2.51 mg/L (mean measured concentration).

Executive summary:

In a 96 -hour fish study, the acute toxicity of Vetimoss was determined according to OECD 203 TG. The 96-hour LC50 of Vetimoss in fish is 2.51 mg/L (mean measured concentration).

Description of key information

In a 96 -hour fish study, the acute toxicity of Vetimoss was determined according to OECD 203 TG. The 96-hour LC50 of Vetimoss in fish is 2.51 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.51 mg/L