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Ecotoxicological information

Long-term toxicity to fish

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Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 April 2017 to XXXX
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Octadecyl dihydrogen phosphate
EC Number:
220-983-1
EC Name:
Octadecyl dihydrogen phosphate
Cas Number:
2958-09-0
Molecular formula:
C18H39O4P
IUPAC Name:
octadecyl dihydrogen phosphate
Constituent 2
Chemical structure
Reference substance name:
Hexadecyl dihydrogen phosphate
EC Number:
222-581-1
EC Name:
Hexadecyl dihydrogen phosphate
Cas Number:
3539-43-3
Molecular formula:
C16H35O4P
IUPAC Name:
hexadecyl dihydrogen phosphate
Constituent 3
Chemical structure
Reference substance name:
Dioctadecyl hydrogen phosphate
EC Number:
221-237-8
EC Name:
Dioctadecyl hydrogen phosphate
Cas Number:
3037-89-6
Molecular formula:
C36H75O4P
IUPAC Name:
dioctadecyl hydrogen phosphate
Constituent 4
Chemical structure
Reference substance name:
Dihexadecyl hydrogen phosphate
EC Number:
218-594-7
EC Name:
Dihexadecyl hydrogen phosphate
Cas Number:
2197-63-9
Molecular formula:
C32H67O4P
IUPAC Name:
dihexadecyl hydrogen phosphate
Constituent 5
Chemical structure
Reference substance name:
hexadecyl octadecyl hydrogen phosphate
Cas Number:
93803-11-3
Molecular formula:
C34H71O4P
IUPAC Name:
hexadecyl octadecyl hydrogen phosphate
impurity 1
Reference substance name:
Unidentified impurities, likely the C16 and C18, and mixed C16/C18 trialkyl phosphates based 31P NMR chemical shift.
Molecular formula:
N/A
IUPAC Name:
Unidentified impurities, likely the C16 and C18, and mixed C16/C18 trialkyl phosphates based 31P NMR chemical shift.
Test material form:
solid
Details on test material:
CAS Number 39471-52-8
Specific details on test material used for the study:
Physical State/Appearance: White waxy solid
Purity: 100%, UVCB substance
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and the 100 mg/L loading rate WAF test group from the freshly prepared bulk test preparation on Days 0, 4, 8, 13, 18, 25 and 32 and from the old (expired) media on Days 1, 6, 11, 15, 20, 27 and 33 (Replicates R1 to R4 pooled) for quantitative analysis.

Duplicate samples were taken 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 material the test medium was prepared as a Water Accommodated Fraction (WAF) of the test material. Based on results from a mixing period trial conducted by Envigo, a 95-hour stirring period followed by a 1-hour settlement period was employed.

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
The test was carried out using freshly fertilized eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were supplied from Osage Catfisheries, and maintained in dechlorinated tap water with an activated carbon and biological filtration system.

The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the seven days preceding the start of the test, the water temperature was controlled at 24 deg.C to 25 deg.C with a dissolved oxygen content of greater than or equal to 8.5 mg O2/L. The breeding stock fish were fed ZM 400 flake food daily and frozen brine shrimp.

Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 21 September 2017 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d

Test conditions

Hardness:
Approximately 140 mg/L as CaCO3
Test temperature:
23 deg.C to 26 deg.C
pH:
6.4 to 8.4
Dissolved oxygen:
At least 4.3 mg/L
Nominal and measured concentrations:
Based on the results of the range-finding test, which was conducted using nominal loading rates of 1.0, 10 and 100 mg/L, the definitive test was conducted at a single nominal loading rate of 100 mg/L.
Details on test conditions:
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25°C with a maximum deviation of +/-1.5°C between test chambers or between successive days, and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods throughout the 33-day exposure period.

The test vessels were aerated via narrow bore glass tubes from Day 13 onwards. The eggs and larvae were not individually identified.

OTHER TEST CONDITIONS
Dissolved oxygen concentrations, water temperature, and pH were recorded before and after each test media renewal. The light intensity was recorded on days when a media renewal took place. The water hardness was measured in the bulk test preparation at the start and in each vessel on termination of the test and was determined.

Surplus food and feces were removed regularly from the test vessels.
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Key result
Duration:
33 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: hatchability, post-hatch survival, growth (length and weight)
Details on results:
Range-Finding Test
There were no differences in hatching, survival or growth between the control and any of the nominal loading rates tested (1.0, 10 and 100 mg/L).

Sub-lethal effects were observed in the control and the 10 and 100 mg/L loading rate WAF test groups. One fish in the control group was observed to have a bent spine from Day 7 to its death on Day 13. On Day 8 of the test, a single fish in the 10 mg/L loading rate WAF test group was observed to be pale and swimming abnormally, and from Day 8 to Day 10, a single fish in the 100 mg/L was observed to be pale.

Definitive Test
The number of dead eggs was low throughout the test.

The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 6 of the test. No significant mortalities or sub-lethal effects of exposure were observed.

Mean hatching rate in the control and 100 mg/L loading rate groups was 75 and 86%, respectively. Mean post-hatch survival rate in the control and 100 mg/L loading rate groups was 99% and 97%, respectively.

The group mean length and weight in the control group were 16.43 mm and 81.5 mg, respectively. The group mean length and weight in the 100 mg/L loading rate group were 17.02 mm and 88.2 mg, respectively.

One larvae in the control group was observed to have a bent spine from Day 8 to Day 12. One larvae in the 100 mg/L loading rate test group was observed to be small on Day 14.

These observations are considered to be due to naturally occurring defects occurring in a the test organism population, and were not attributable to the test material.
Reported statistics and error estimates:
Analysis of the number of eggs successfully hatching from the control and the 100 mg/L loading rate WAF test group were compared using the Chi squared 2x2 Table test. No significant differences (P=0.05) in hatching success were found between the control and the 100 mg/L loading rate WAF test group.

Analysis of the fish length data obtained at termination of the test from the control and the 100 mg/L loading rate WAF test group were compared using a Students t-test for homogenous Variances. No significant differences (P=0.05) in larval length were found between the control and the 100 mg/L loading rate WAF test group using the above method of statistical analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The exposure of newly fertilized eggs of fathead minnows to the test material was considered to have no effect on the hatching success or the survival and growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

A study was performed to assess the effects of the test material on freshly hatched larvae of the fathead minnow (Pimephales promelas).

Due to the low aqueous solubility and complex nature of the test material, the test solutions were prepared as a Water Accommodated Fraction (WAF).

Based on the results of a preliminary range-finding test, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a WAF of the test material for a period of 33 days at a temperature of 23°C to 26°C under semi-static test conditions. The test solutions were renewed three times per week throughout the test. The test material solution was prepared by stirring a nominal loading rate of test material (100 mg/L) in test water using a magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface for 95 hours. After the stirring period the mixture was allowed to stand for 1 hour before the aqueous phase or WAF was removed by mid-depth siphoning to give the 100 mg/L loading rate WAF.

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

Chemical analysis of the freshly prepared test preparations on Days 0, 4, 8, 13, 18, 25 and 32 and of the old (expired) media on Days 1, 6, 11, 15, 20, 27 and 33 showed measured test concentrations of less than the limit of quantification. 

The dissolved fraction of the WAF test solution may have been comprised of one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Over the duration of the test, there were no significant mortalities or sub-lethal effects resulting from the exposure of fathead minnow (Pimephales promelas) larvae to the test material at a nominal loading rate of 100 mg/L.

The exposure of newly fertilized eggs of fathead minnows to the test material was considered to have no effect on the hatching success or the survival and growth of the larvae. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.