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Ecotoxicological information

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 22 November 2016 and 22 March 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
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)
EC Number:
300-947-2
EC Name:
O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)
Cas Number:
93964-99-9
Molecular formula:
C8H19N.C8H19O3PS
IUPAC Name:
octan-1-amine dibutyl sulfanylphosphonate
impurity 1
Chemical structure
Reference substance name:
Octylamine
EC Number:
203-916-0
EC Name:
Octylamine
Cas Number:
111-86-4
Molecular formula:
C8H19N
IUPAC Name:
octan-1-amine
Test material form:
liquid
Specific details on test material used for the study:
Identification: X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9
Nature of Test Item: UVCB
Physical state/Appearance: Amber colored viscous liquid
Batch: X-019575-00-00
Purity: Not applicable (100% as UVCB)
Expiry Date: 01 June 2017
Storage Conditions: Room temperature in darkness

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. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive test
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates (replicates R1 – R4) at 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
The Determination of General Physico-Chemical Properties study performed on the test item (Envigo Study Number BT67DH) indicated that at initial loading rates of 10 and 100 g/L the water solubility was greater than 4 g/L. However preliminary work performed for the aquatic ecotoxicology tests showed that under test conditions it was not possible to obtain a true solution of the test item at a nominal concentration of 100 mg/L. Therefore, due to the limited aqueous solubility under test conditions 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.

Range-finding test
Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 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.

Initial experiments
1+2) Nominal amounts of test item (18.75, 37.5, 75, 150 and 300 mg) were each separately added to the surface of 3 liters of test water to give the 6.25, 12.5, 25, 50 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

3) Amounts of test item (29.8, 55.9, 96.4, 167.6 and 299.7 mg) were each separately added to the surface of 3 liters of test water to give the nominal 10, 18, 32, 56 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

Definitive test
Nominal amounts of test item (30, 54, 96, 168 and 300 mg) were each separately added to the surface of 3 liters of test water to give the 10, 18, 32, 56 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
Dilutions were made from the 32 and 56 mg/L loading rate WAFs to give the 3.2 and 5.6 mg/L loading rates, respectively. Due to the relatively low test concentrations, it was not practical to prepare the 3.2 and 5.6 mg/L loading rates as individual WAFs and dilutions were required.

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 maintaining the water temperature at 18 °C to 22 °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:
21 - 22 ºC
pH:
7.6 - 7.9
Dissolved oxygen:
7.9 - 8.9 mg O2/L
Nominal and measured concentrations:
Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 3.10 to 90.0 mg/L in the 3.2 to 100 mg/L loading rate WAFs. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 2.91 to 93.4 mg/L.
Analysis was based on measurement of the dibutylthiophosphoric acid component of the test item. A reference material was not analyzed to confirm the compound 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 dibutylthiophosphoric acid 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.

Range-finding test (nominal): 1.0, 10 and 100 mg/L
Initial experiment 1+2 (nominal): 6.25, 12.5, 25, 50 and 100 mg/L
Initial experiment 3 (nominal): 10, 18, 32, 56 and 100 mg/L
Definitive test (nominal): 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L


Details on test conditions:
In the range-finding test, 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °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 daphnids was recorded.
The control group was prepared using the WAF methodology and maintained under identical conditions, but not exposed to the test item.

Definitive test
As in the range-finding 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 maintaining the water temperature at 18 °C to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (between 200 and 1200 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 prepared using the WAF methodology and maintained under identical conditions, but not exposed to the test item.
The test preparations were not renewed during the exposure period.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EL50
Remarks:
loading Rate WAF
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Remarks:
loading rate WAF
Effect conc.:
18 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, immobilization was observed at 10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the initial experiment.
Chemical analysis of the10 and 100 mg/L loading rate WAFs at 0 hours showed measured test concentrations of 12.4 and 96.7 mg/L, respectively. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Initial Experiments
In the first experiment, low levels of immobilization were observed in the 6.25 and 12.5 mg/L loading rate WAF of 25% and 5%, respectively. The 25 mg/L loading rate WAF had 90% immobilization, and the 50 and 100 mg/L loading rate WAF test groups had 100% immobilization. The measured concentrations in the 6.25 and 12.5 mg/L loading rate WAF test groups were inconsistent with dosing.
In the second experiment, 5% immobilization was observed in the control, 6.25 and 25 mg/L loading rate WAFs. The 12.5 mg/L loading rate WAF group had 10% immobilization. The 50 and 100 mg/L loading rate WAF test groups had 100% immobilization. The measured concentrations in the 6.25 mg/L loading rate WAF were inconsistent with dosing.
In the third experiment, there was a control failure due to 25% sub-lethal effects due to daphnia covered in debris and trapped at the surface. Additionally, there were sub-lethal effects and immobilization observed in all test groups.
The loading rates in the definitive test were adjusted according to these results.

Definitive Test

Sub-Lethal Effects
A sub-lethal effect of exposure was observed in the 32 and 56 mg/L loading rate WAF test groups. This response was reduced mobility.

Validation Criteria
The test was considered to be valid given that none 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 greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
Temperature was maintained at 21 ºC to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 389 to 415 lux.

Vortex Depth Measurements
The vortex depths were recorded at the start and end of the mixing period, and were observed to be approximately 1% of the water column height.

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, all loading rates were observed to be a clear, colorless water column with an oily layer of orange test item floating on the surface.
After 23 hours stirring and a 1-Hour standing period, all loading rates were observed to be a clear, colorless water column with an orange/brown oily dispersions floating on the surface. These observations confirmed the limited solubility of the test item under test conditions. 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 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L loading rates were observed to be clear, colorless solutions.

Results with reference substance (positive control):
A positive control (Envigo Study Number XT01HS, performed 20 October 2016 to 22 October 2016) 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 was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All analysis was carried out using the ToxRat Professional computer software package (ToxRat).

24hrs EC50 (mg/L): 1.3 (1.0 - 1.8, 95% Confidence Limits (mg/L))
24 hrs No Observed Effect Concentration (NOEC) (mg/L): 1.0
24 hrs Lowest Observed Effect Concentration (LOEC) (mg/L): 1.8

48 hrs EC50 (mg/L): 1.2 (1.1 - 1.3, 95% Confidence Limits (mg/L))
48 hrs No Observed Effect Concentration (NOEC) (mg/L): 0.56
48 hrs Lowest Observed Effect Concentration (LOEC) (mg/L): 1.0

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Immobilization Data

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal loading rates gave the following results:

Time

(hours)

EL50

(mg/L Loading Rate WAF)

95% Confidence limits
(mg/L Loading Rate WAF)

24

32

27  -  36

48

31

Not determined*

*The 95% confidence limits were notdetermined due to mathematical reasons.

The No Observed Effect Loading rate after 24 and 48 hours exposure was 18 mg/L loading rate WAF.  Correspondingly, the Lowest Effect Loading rate was considered to be 32 mg/L loading rate WAF.  

The slopes and their standard errors of the response curves at 24 and 48 hours were 8.0 (SE = 0.029) and 20 (SE = 0.0575), respectively.  

Validation of Mixing Period

Preliminary investigational work indicated that there was no significant 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results based on nominal concentrations:
EL50 48hrs: 31 mg/L Loading Rate WAF
NOEL 48hrs: 18 mg/L
LOEL 48 hrs: 32 mg/L

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

Preliminary solubility work performed indicated that under test conditions the test item was not readily soluble. Therefore, due to the limited aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test and initial experiments, 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 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21°C to 22 °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 was based on measurement of the dibutylthiophosphoric acid component of the test item. No reference material was analyzed to confirm the compound measured.

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 3.10 to 90.0 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 2.91 to 93.4 mg/L.

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 dibutylthiophosphoric acid 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.


  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 Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/L)

Lowest Observed Effect Loading Rate (LOEL) (mg/L)

48

31

Not determined

18

32

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