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Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 January 2014 - 15 Aprl 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Not considered to impact the integrity or outcome of the study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
Not considered to impact the integrity or outcome of the study
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine
EC Number:
939-981-3
IUPAC Name:
Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10, 32 and 100 mg/L loading rate WAF

- Sampling method: The test item concentration in the test samples was monitored by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard. Using this method the test item gave a chromatographic profile consisting of two unresolved peaks. The species that was monitored had a m/z of 108 corresponding to various alkyl pyridines which are present in the test item. However, this was confirmed by the m/z only, and not by comparison with a reference standard. In the absence of a reference standard, the test item was used to prepare analytical standards and hence measured concentrations of the 108 m/z ion were relative to the concentration of this species in the test item.

- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
Nominal amounts of test item (5.0, 16, 50, 160 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 3.2, 10, 32 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. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that the filtration had removed all of the microparticles of test item in the water column.

- Controls:
A solvent control was prepared as above by dispensing a nominal amount (100 mg) of propylene glycol onto the surface of 5 liters of test media. The solvent control did not require filtration through a glass wool plug at the end of stirring.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
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 and 3 .2 mg/L loading rates were observed to be clear colorless water columns with test item floating at the surface. The 10 and 32 mg/L loading rates were observed to be clear colorless water columns with some test item floating at the surface, suspended throughout and at the bottom of the vessel. The 100 mg/L loading rate was observed to be a slightly brown clear water column with test item at the surface, suspended throughout and at the bottom of the vessel. After 23 hours stirring and a I-Hour standing period the 1.0 and 3.2 mg/L loading rates were observed to be clear colorless water columns with test item floating at the surface. The 10 mg/L loading rate was observed to be a clear colorless water column with test item floating at the surface and settled at the bottom of the vessel. The 32 mg/L loading rate was observed to be a very pale brown water column with test item at the surface, on the bottom of the vessel and suspended throughout. The 100 mg/L loading rate was observed to be a brown colored water column with test item floating at the surface, on the bottom of the vessel and suspended throughout. Examination of the WAFs showed that undissolved test item was visible in the water columns and therefore it was considered justifiable to remove the W AFs by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of the undissolved test item. During the test the 1.0, 3.2 and 10 mg/L loading rates were observed to be clear, colorless solutions, the 32 mg/L loading rate was observed to be a slightly brown clear solution and the 100 mg/L loading rate was observed to be a brown clear solution.

Solubility:
The test item concentration in the test samples was monitored by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard. Using this method the test item gave a chromatographic profile consisting of two unresolved peaks. The species that was monitored had a m/z of 108 corresponding to alkyl pyridines which were present in the test item. However, this was confirmed by the m/z only, and not by comparison with a reference standard. In the absence of a reference standard, the test item was used to prepare analytical standards and hence measured concentrations of the 108 m/z ion were relative to the concentration of this species in the test item. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of the 108 m/z ion of between 87% and 118% of the concentration present in the test item. These results indicated that the 108 m/z ion was readily soluble in the test medium and stable over the period of test medium renewal, however observations performed during the preparation of the WAFs confirmed that a significant amount of undissolved test item still remained. Given that toxicity cannot be attributed to a single component or mixture of components, but to the test item as a whole, and that the analytical method employed was specific to only the 108 m/z species present in the test item and not the test item as a whole, the results were based on nominal test concentrations alone.

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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaC03.
Test temperature:
20°C
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
Dissolved oxygen:
Aerated until the dissolved oxygen concentration was approximately air-saturation value
Nominal and measured concentrations:
Nominal loading rates of 1, 3.2 10, 32 and 100 mg /L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass jar
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL
- Volume of solution: 200 mL
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ELGA Pure Lab Option R-15 water purification system. Water, Fisher Scientific, LC-MS grade
- Culture medium different from test medium: Elendt M7 medium
- Intervals of water quality measurement: start and termination of the test

OTHER TEST CONDITIONS
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: 542 to 628 lux

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to nominal loading rates of 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: After 24 and 48 hours the number of immobilized Daphnia magna were recorded.
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Volume of solution:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
A positive control (Harlan Study Number 41301833) used potassium dichromate as the reference item. The positive control was conducted between 16 May 2013 and 18 May 2013

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
57 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Remarks:
WAF
Basis for effect:
mobility
Details on results:
Immobilisation of control: 0
Results with reference substance (positive control):
Historical control data - 48 hr EC50 was 0.71 mg/L (Harlan Study Number 41301833 used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L)
Reported statistics and error estimates:
The EL50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999) and at 24 hours were calculated using the geometric mean

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 a 48 hr EL50 of 48 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, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of approximately 20 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

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 test concentrations alone which gave an 48 hr EL50 of 48 mg/L Loading Rate WAF