Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 04 May 2017 and 20 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Identification: X-19574 Amines
Physical state/Appearance: Amber colored viscous liquid
Batch: X-19574-00-00
Purity: 100% UVCB
Expiry Date: 01 June 2017
Storage Conditions: Room temperature, in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 0.028 and 1.0 mg/L loading rate WAF test preparations at 0 and 72 hours from fresh media and from the control and 0.028 mg/L loading rate WAF test vessels at 24 and 96 hours from old media for quantitative analysis. The 0, 24 and 72 hour samples were stored frozen prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. The EL50 value obtained from the Algal Growth Inhibition study (Envigo Study Number NM42GB) was determined to be 0.028 mg/L loading rate WAF and was the lowest of these two EL50 values and hence the test was conducted at a single loading rate of 0.028 mg/L
A nominal amount of test item (28.4 µL, equivalent to approximately 22 mg) was added to the surface of 22 liters of test water to give a 1.0 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give a 1.0 mg/L loading rate WAF. An aliquot (560 mL) of the 1.0 mg/L loading rate WAF was diluted to a final volume of 20 liters with test water to give the 0.028 mg/L loading rate WAF.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 15 March 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 01 May 2017 to 08 May 2017. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at 14 °C to 15 °C with a dissolved oxygen content of greater than or equal to 9.7 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.5 cm (sd = 0.37) and a mean weight of 1.47 g (sd = 0.26) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.51 g bodyweight/liter.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Test temperature:
14 -15 ºC
pH:
7.9 - 8.1
Dissolved oxygen:
8.5 - 10.5
Nominal and measured concentrations:
Chemical analysis of the 0.028 mg/L fresh test preparations at 0 and 72 hours showed measured test concentrations to be 0.013 mg/L and 0.017 mg/L, respectively. Chemical analysis of the old test preparations at 24 and 96 hours showed measured test concentrations to be 0.0062 mg/L and 0.0085 mg/L, respectively. Chemical analysis was also conducted on the 1.0 mg/L loading rate WAF from which the 0.028 mg/L loading rate WAF test solution prepared at 0 and 72 hours showed measured test concentrations to be
0.46 mg/L to 0.54 mg/L, respectively.
The ion that was monitored by positive mode LC-MS had a m/z of 268. This mass corresponds with the [M+H]+ ion of an unsaturated C18 amine (C18H37N). A reference material was not analyzed to confirm the compound being measured.
The test item concentrations were based on the amount of test item weighed out to prepare the standards. No correction of the test item concentration for the content of the amine component was undertaken. Whilst only the amine component of the test item was measured, the concentration of the standards was in terms of test item (mg/L), as such the calculated values for the test solutions are in the same term (mg/L test item).
As the test item is UVCB in nature, the dissolved portion may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Nominal loading rate WAF - 0.028 mg/L
Details on test conditions:
Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 0.028 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
0.028 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Mortality Data
There were no mortalities in 7 fish exposed to a 0.028 mg/L loading rate WAF for a period of 96 hours. The No Observed Effect Loading rate (NOEL) was considered to be 0.028 mg/L loading rate WAF, correspondingly the Lowest Observed Effect Loading rate (LOEL) was not determined.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a 0.028 mg/L loading rate WAF for a period of 96 hours.

Validation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.

Any other information on results incl. tables

 Validation of Mixing Period

Preliminary investigational work indicated that there was a significant increase in the amount of dissolved test item when the preparation period was extended from 24 to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

Inspection of the mortality data gave the following results:

Time (h)

LL50

(mg/L Loading Rate WAF)

1

>0.028

3

>0.028

6

>0.028

24

>0.028

48

>0.028

72

>0.028

96

>0.028

Water Quality Criteria

Temperature was maintained at 14 °C to 15 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex Depth Measurements

The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAF.

At the start of the mixing period the 1.0 mg/L loading rate was observed to be a clear colorless water column with oily droplet/globules of test item at the water surface. After
95 hours stirring and a 1-Hour standing period the 1.0 mg/L loading rate was observed to be a clear colorless water column with test item at the water surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the control and 0.028 mg/L loading rate was observed to be a clear, colorless solution.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated using the threshold approach and gave a 96-Hour LL50 value of greater than 0.028 mg/L loading rate WAF. The No Observed Effect Loading rate was 0.028 mg/L loading rate WAF.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods…….

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EL50value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate.  If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50is greater than the threshold loading rate. The EL50value obtained from the Algal Growth Inhibition study (Envigo Study Number NM42GB) was determined to be 0.028 mg/L loading rate WAF and was the lowest of these two EL50values and hence the test was conducted at a single loading rate of 0.028 mg/L.

Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 0.028 mg/L for a period of 96 hours at a temperature of 14°C to 15 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis was based on measurement of an unsaturated C18 amine component of the test item. No reference item was analyzed to confirm the component measured.

Chemical analysis of the 0.028 mg/L fresh test preparations at 0 and 72 hours showed measured test concentrations to be 0.013 mg/L and 0.017 mg/L, respectively. Chemical analysis of the old test preparations at 24 and 96 hours showed measured test concentrations to be 0.0062 mg/L and 0.0085 mg/L, respectively. Chemical analysis was also conducted on the 1.0 mg/L loading rate WAF from which the 0.028 mg/L loading rate WAF test solution preparedat 0 and 72 hours showed measured test concentrations to be 0.46 mg/L and 0.54 mg/L, respectively.

As the test item is UVCB in nature, the dissolved portion may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components, but to the test item as a whole, the results are based on the nominal loading rate only.

Exposure of rainbow trout to the test item gave LL50values of greater than 0.028 mg/L loading rate WAF. The No Observed Effect Loading Rate was 0.028 mg/L loading rate WAF.