Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
Batch/Lot number: 0002293298
Appearance: High-viscous, colourless to yellowish liquid
Purity: Considered as 100%
Expiry date: 22 November 2019
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to assure personnel health and safety.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test item was measured at the test concentration at the start and at the end of each renewal period. Sample from the control was taken for analysis at least at the beginning of each renewal period.

Test solutions

Vehicle:
no
Details on test solutions:
A stock solution with a concentration of 1000 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (aquarium water) using ultrasonic bath (approximately 10 minutes). The test solutions were prepared by appropriate dilutions of this stock solution just before introduction of the fish.
Because the test item is not stable over the 96-hour exposure period (based on the analytical method validation, Citoxlab Study Codes: 18/102-316AN and 18/102-316ANA), the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test item solutions were prepared by the method described above.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
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 each concentration and control
Body length of animals: 2.0 ± 1.0 cm
Acclimatisation: at least 12 days
Animal health: Fish are bred in a standard commercial fish farm, under disease- and parasite-controlled conditions.
Holding water: Circulated and filtered tap water (same as that will be used for the test)
Food and Feeding: The fish will not be fed during the test.
Water: Circulated and filtered water will be used for holding of fish. The fish will be held during the test in water of the same quality used for holding.
Source of water: Circulated and filtered human drinking water. Filtration is performed by carbon filter. Quality control analysis is performed once every three months and microbiological assessment is performed monthly.
Light: 12 to 16 hours photoperiod daily.
Temperature range: 20 – 24°C with a maximum deviation of ± 1°C
Dissolved oxygen concentration: At least 80 per cent of air saturation
pH values: 6.0 to 8.5
Feeding: Three times per week or daily until 24 hours before the start of the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observation

Test conditions

Hardness:
151 mg/L (as CaCO3)
Test temperature:
20.7 - 22.1 °C
pH:
7.93 - 8.32
Dissolved oxygen:
77-100 % of air saturation value
Salinity:
Fresh Water
Nominal and measured concentrations:
The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25, 50 and 100 mg/L.

The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods.
The corresponding measured geometric mean test item concentrations were: 6.13; 11.08; 23.07; 47.41 and 99.03 mg/L.
Details on test conditions:
The light-dark cycle during the test will be 16 hours light (artificial illumination) and 8 hours darkness.
Reference substance (positive control):
not required

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
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
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
ca. 25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
ca. 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Details on results:
MORTALITY DATA

Cumulative mortality data from the exposure of zebrafish to the test material during the definitive test are given below in Table 4.
OBSERVATIONS

Behaviour of fish was found as normal in the control group and at the tested concentration range of 6.25 - 25 mg/L (nominal).

Localization close to the surface of the water was observed at the nominal concentration of 50 mg/L 24 hours after the start of the test.

At the test group of 100 mg/L (nominal) localization at the bottom of aquarium or close to the surface of the water was observed during the experiment 24 hours after the start of the test and thereafter.

Any other information on results incl. tables

Mortality

Table 4:Cumulative mortality data in the Definitive Test

Concentration

Cumulative mortality[number of fish]

(initial population = 7 fish)

Nominal
(mg/L)

3h

6h

24h

48h

72h

96h

Control

0

0

0

0

0

0

6.25

0

0

0

0

0

0

12.5

0

0

0

0

0

0

25

0

0

0

0

0

0

50

0

0

0

0

0

0

100

0

0

0

0

0

1

Body Weight

 

The body weight of 7 fish was weighed at the start of the test. The measured and calculated data are listed below in Table 5.

 

Table 5: Measured and calculated data of bodyweight

Concentration

Measured weight
of 7 fish (g)

Calculated mean
weight of 1 fish (g)

Loading of testing aquarium
(g fish/
Ltesting liquid)

Nominal
(mg/L)

Control

0.98

0.14

0.20

6.25

1.09

0.16

0.22

12.5

1.06

0.15

0.21

25

1.21

0.17

0.24

50

1.15

0.16

0.23

100

1.23

0.18

0.25

 

There was no considerable difference observed concerning body weights between the groups.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system.

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine were the following:

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L (nominal)
The 96h LC100 value: > 100 mg/L (nominal)
The 96h No-Observed Effect Concentration (NOEC): 25 mg/L (nominal)
The 96h Lowest Observed Effect Concentration (LOEC): 50 mg/L (nominal)
Executive summary:

The acute toxicity ofthe test itemwas assessed with acute fish toxicity test onZebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system.

 

Concentrations were selected based on the results of a preliminary experiment. Because significant toxicity was observed at the examined concentration levels during the preliminary range-finding test,five test concentrations in a geometric serieswith a separation factor of 2.0and one untreated control group were tested in the main experiment under semi-static conditions.

 

The nominal loading rates of test item used in the main experiment were 6.25, 12.5, 25, 50 and 100 mg/L.

 

The test concentrationswereanalytically determined at the start and at the end of the first and at the last renewal periods.The corresponding measured geometric mean test item concentrations were: 6.13; 11.08; 23.07; 47.41 and 99.03 mg/L.

As the measured concentrations deviated not more than 20% from the nominal concentrations (except of the end sample of the last renewal period at 12.5 mg/L concentration), biological results are based on thenominal loading rates of test item.

 

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litre test solution.

 

The24, 48, 72 and 96 hours LC50values of the test item, the NOEC, LOEC and LC100values were determined from the raw data.

 

All validity criteria were met during this study.

 

Under the conditions of this acute fish toxicity studyon Zebrafish (Brachydanio rerio)the observed and calculated endpoints for the effect ofEthanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanaminewere the following:

 

The 24h, 48h, 72h and 96h LC50value: > 100 mg/L (nominal)

The 96h LC100value: > 100 mg/L (nominal)

The 96h No-Observed Effect Concentration (NOEC): 25 mg/L (nominal)

The 96h Lowest Observed Effect Concentration (LOEC): 50 mg/L (nominal)