Registration Dossier

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:
This study was conducted between 06 October 20147 and 16March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid: viscous
Details on test material:
Identification:
1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Appearance/Physical state:
clear, colorless, viscous liquid
Batch:
WA 1508
Purity:
100% UVCB
Expiry date:
01 January 2021
Storage conditions:
room temperature, in the dark
Specific details on test material used for the study:
Identification: 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Batch: WA 1508
Purity: not supplied
Physical state/Appearance: clear colorless viscous liquid
Expiry Date: 01 January 2021
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-Finding Tests
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
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours

Test solutions

Vehicle:
yes
Remarks:
Reconstituted water (Elendt M7 medium)
Details on test solutions:
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 (5, 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 95 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 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

Definitive Test
Nominal amounts of test item (5, 9, 16, 28 and 50 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 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 95 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 to 100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 1) in a temperature controlled room maintaining the water temperature at 18 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
Remarks:
Elendt M7 medium
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
18-22 ”C
pH:
The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
The dissolved test item 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.
Details on test conditions:
Experimental Design and Study Conduct

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 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 (5, 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 95 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 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.
In the range finding test five 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. Two replicate test and control vessels were prepared. 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 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. Only concentrations within the range to be used for the definitive test were analyzed.

Initial Experiment
Based on the results of the range-finding test and initial experiment was conducted at concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAF.
Significant immobilization was observed throughout all of the test concentrations. This was not in line with what was seen in the range finding tests and no NOEL value could be determined. Therefore the test was repeated.

Definitive Test
Nominal amounts of test item (5, 9, 16, 28 and 50 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 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 95 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 to 100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours.

Exposure Conditions
As in the range finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five 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 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 maintained under identical conditions but not exposed to the test item.
Semi static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 Hour old test media into the fresh test media.

Assessments
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 within 15 seconds after gentle agitation.

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

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 freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary. Samples of the 10 mg/L loading rate WAF were not provided for analysis in error.
.
Data Evaluation
Statistical Analysis
The EL50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Logit analysis using Linear Maximum Likelihood regression. The LOEL and the NOEL at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
• The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

Reference substance (positive control):
yes
Remarks:
Potassiium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
3.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
Range-finding Test
No sub lethal effects of exposure were observed throughout the test.
No immobilization was observed at 1.0 mg/L loading rate WAF, however, immobilization was observed in the control group and at 10 and 100 mg/L loading rate WAFs.
Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the definitive test.
Chemical analysis of the 1.0 and 10 mg/L loading rate WAF test preparations at 0 hours (see Annex 4) showed measured test concentrations of less than the LOQ of the analytical method employed determined to be 0.059 mg/L, and 5.2 mg/L were obtained respectively. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions.

Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.38 to 6.0 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.25 to 4.4 mg/L.
The dissolved test item 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.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

Analysis of the immobilization data by Logit analysis using Linear Maximum Likelihood regression at 24 and 48 hours based on the nominal loading rates gave the following results:
Time(Hour) EL50 (mg/L Loading Rate WAF) 95% Confidence limits(mg/L Loading Rate WAF)
24 11 Not determined
48 3.9 Not determined
The NOEL rate after 48 hours exposure was 3.2 mg/L loading rate WAF. Correspondingly the LOEL rate was considered to be 5.6 mg/L loading rate WAF.
The slopes and their standard errors of the response curves at 24 and 48 hours were 44 (standard error = 0.012) and 19 (standard error = 0.028) respectively.

Sub-Lethal Effects
No sub lethal effects of exposure were observed throughout the test.

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 equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
Temperature was maintained at 21 °C 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 712 to 819 lux.

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 all loading rates were observed to be clear colorless water columns with test item or globules of test item floating on the surface. After 95 hours stirring and a 1 Hour standing period all loading rates were observed to be clear colorless water columns with test item or a slick of test item at the surface of the water. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present.
At the start and throughout the test all control and test solutions were observed to be a clear colorless solution.
Results with reference substance (positive control):
A positive control (Envigo study number LK67NP) 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 Trimmed Spearman Karber method at 24 hours and the Binomial Distribution method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
Time Point(Hours) EC50(mg/L) 95% Confidence Limits (mg/L) NOEC(mg/L) LOEC (mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

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

Any other information on results incl. tables

Table 2: Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5N

5 N

5 N

5 N

1.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.8

0

0

0

0

0

0

5 N

5 N

5 N

5 N

3.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

5.6

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

1

2

2

5

25

5 N

4 N

3 N

3 N

 

Nominal Loading Rate
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.8

0

0

0

1

1

5

5 N

5 N

5 N

4 N

3.2

1

0

0

0

1

5

4 N

5 N

5 N

5 N

5.6

5

5

5

5

20

100

AI

AI

AI

AI

10

5

5

5

5

20

100

AI

AI

AI

AI

R   = Replicate

N     = Normal

AI  =All daphnia immobilized

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
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 equal to or greater than 3 mg/L in the control and test vessels
Conclusions:
Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:
Time Point(Hours) EL50(mg/L Loading Rate WAF) 95% Confidence Limits (mg/L Loading Rate WAF) NOEL(mg/L) LOEL (mg/L)
48 3.9 Not determined 3.2 5.6

Executive summary:

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., 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 and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to WAFs of the test item over a range of nominal loading rates of1.0,1.8,3.2,5.6mg/L and10 mg/L for 48 hours at a temperature of 21 °C to 22 °C under semi‑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 1.0, 1.8, 3.2 and 5.6 mg/L loading rate WAF fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.38 to 6.0 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from 0.25 to 4.4 mg/L.

The dissolved test item 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 ofDaphnia 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)

NOEL
(mg/L)

LOEL
(mg/L)

48

3.9

Not determined

3.2

5.6

NOEL   = No Observed Effect Loading rate

LOEL   = Lowest Observed Effect Loading rate