Amines, C12-14-alkyl, isooctyl phosphates

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 01 December 2014 and 16 February 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-14-tert-alkyl, isooctyl mono phosphates

IUPAC Name:

Amines, C12-14-tert-alkyl, isooctyl mono phosphates

Constituent 2

Reference substance name:

Unknown, including minor amounts of diphosphate species, water. Possibly residual unreacted starting material and unknown byproducts.

IUPAC Name:

Unknown, including minor amounts of diphosphate species, water. Possibly residual unreacted starting material and unknown byproducts.

Test material form:

liquid: viscous

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding test
A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding Test
Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2.0 and 2.0 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. 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 23 hours and the mixtures 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 WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

Definitive Test
Nominal amounts of test item (10, 25, 64, 160 and 400 mg) were each separately added to the surface of 10 liters of test water to give the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rates respectively. 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 23 hours and the mixtures 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 WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rate WAFs

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 21 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

Water temperature was recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer. Temperature was maintained at approximately 21 ºC throughout the test.

pH:

pH was recorded at the start and termination of the test using a Hach Flexi handheld meter. There were no treatment related differences.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test using a Hach Flexi handheld meter. There were no treatment related differences.

Nominal and measured concentrations:

Range-finding test: Nominal loading rates of 1.0, 10 and 100 mg/L.
Definitive test: Nominal loading rates of 1.0, 2.5, 6.4, 16 and 40 mg/L

Details on test conditions:

Test Water
Reconstituted water (ISO medium) used for both the range-finding and definitive tests.


Procedure
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.


Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.


Range-finding Test
The loading rates to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.

Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2.0 and 2.0 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. 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 23 hours and the mixtures 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 WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 2.5, 6.4, 16 and 40 mg/L.


Experimental Preparation
Nominal amounts of test item (10, 25, 64, 160 and 400 mg) were each separately added to the surface of 10 liters of test water to give the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rates respectively. 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 23 hours and the mixtures 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 WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rate WAFs.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Exposure Conditions
In the definitive test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light (502 to 538 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period.


Evaluations
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.


Chemical Analysis of Test Loading Rates
Samples were taken from the control and each test group from the bulk test preparation at
0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits of 14 - 17 mg/L loading rate WAF

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at 1.0 mg/L loading rate WAF. However, 40% immobilization was observed at 10 mg/L loading rate WAF and 100% immobilization was observed at 100 mg/L loading rate WAF.

Based on this information loading rates of 1.0, 2.5, 6.4, 16 and 40 mg/L were selected for the definitive test.

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.98 mg/L to 53 mg/L. Chemical analysis of the old test preparations at 48 hours showed measured test concentrations to range from 0.82 mg/L to 68 mg/L indicating that the test item was stable under test conditions.

Definitive Test
The 48 hour EL50 was 17 mg/L loading rate WAF with 95 % confidence limits of 14 - 17 mg/L loading rate WAF.
The No Observed Effect Loading rates after 24 and 48 hours exposure were 16 and 6.4 mg/L loading rate WAF respectively. Correspondingly the Lowest Effect Loading rates were considered to be 40 and 16 mg/L loading rate WAF.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41400711) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al. 1977 ) at 48 hours based on the nominal test concentrations gave the following results:
The 24 hour EC50 was 0.87 mg/L with 95 % confidence limits of 0.75 - 1.0 mg/L.
The 48 hour EC50 was 0.71 mg/L with 95 % confidence limits of 0.65 - 0.78 mg/L.
The NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L.

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The EL50 values and associated confidence limits at 24 and 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977). The LOEL and the NOEL at 24 and 48 hours were calculated using the Fisher’s Exact Test. All results were calculated using the ToxCalc computer software package (ToxCalc, 1999).

Any other information on results incl. tables

Validation of Mixing Period

Preliminary investigational work indicated that there was no increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Chemical Analysis of Test Loading Rates

Chemical analysis of the fresh test preparations at 0 hours (see Appendix 6) showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0157 mg/L, to 27 mg/L. Chemical analysis of the old test preparations at 48 hours showed measured test concentrations to range from less than the LOQ to 28 mg/L, indicating that the dissolved test item loading rate had been maintained throughout the exposure period.

 

The dissolved test item 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 were based on nominal loading rates only.

Immobilization Data

Time (h)	EL50 (mg/L Loading Rate WAF)	95% Confidence limits (mg/L Loading Rate WAF)
24	23	20 - 26
48	17	14 - 21

Validation Criteria

The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Water Quality Criteria

Temperature was maintained at approximately 21 ºC 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 the 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 WAFs.

 

At the start of the mixing period the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rates were observed to be clear colorless water columns with globules of test item at the water surface. After 23 hours stirring and a 1-Hour standing period the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rates were observed to be clear colorless water columns. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 1.0, 2.5, 6.4, 16 and 40 mg/L loading rates were observed to be clear, colorless solutions.  

See attached background material for results:

Cumulative Immobilization Data in the Range-finding Test

Cumulative Immobilization Data in the Definitive Test

Water Quality Measurements

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EL50 was 17 mg/L loading rate WAF with 95 % confidence limits of 14 - 17 mg/L loading rate WAF.
The No Observed Effect Loading rates after 48 hours exposure was 6.4 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rates was considered to be 16 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 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).

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 2.5, 6.4, 16 and 40 mg/L for 48 hours at a temperature of approximately
21°C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Results

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.0157, mg/L to 27 mg/L. Chemical analysis of the old test preparations at 48 hours showed measured test concentrations to range from less than the LOQ to 28 mg/L, indicating that the dissolved test item concentration had been maintained throughout the exposure period.

 

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.

 

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L LoadingRate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
48	17	14 - 21	6.4	16