Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 July 2017 and 23 September 2017

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EC No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and glycidyl tolyl ether
Physical state/Appearance: Dark yellow viscous liquid
Batch: Ei 4015
Purity: 100% UVCB
Expiry Date: 15 November 2021
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

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 the concentration to be used for the definitive test was analyzed.

Vehicle:

yes

Remarks:

Reconstituted water (ISO 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.
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. 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 once through a glass wool plug (2-4 cm in length) and twice through filter paper. 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 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering showed no microparticles of test item in any of the loading rates, however, the 10 and 100 mg/L loading rates were observed to be slightly cloudy and cloudy dispersions respectively.
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. The 0-Hour samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
Nominal amounts of test item (5.0, 9.0, 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 23 hours and the mixtures allowed to stand for 1-Hour. Observations made on the WAFs indicated that test item was dispersed in the water column of the 10 mg/L loading rate. As a precaution, all of the WAFs were removed 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 75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that no microdispersions of test item were observed in the media.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours

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 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.

Test type:

static

Water media type:

freshwater

Remarks:

Elendt M7 medium

Limit test:

no

Total exposure duration:

48 h

Test temperature:

22 ”C

pH:

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC

Dissolved oxygen:

equal to or greater than 3 mg/L

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

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

Details on test conditions:

Experimental Design and Study Conduct

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 (5.0, 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. 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 once through a glass wool plug (2-4 cm in length) and twice through filter paper. 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 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering showed no microparticles of test item in any of the loading rates, however, the 10 and 100 mg/L loading rates were observed to be slightly cloudy and cloudy dispersions respectively.
In the range-finding test 5 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. The 0-Hour samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
Nominal amounts of test item (5.0, 9.0, 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 23 hours and the mixtures allowed to stand for 1-Hour. Observations made on the WAFs indicated that test item was dispersed in the water column of the 10 mg/L loading rate. As a precaution, all of the WAFs were removed 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
75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that no microdispersions of test item were observed in the media.
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 jars 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
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. 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 loading rate WAF test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates (replicates R1 – R4) at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described above.

Data Evaluation
Statistical Analysis
The EL50 values and associated confidence limits at 24 and 48 hours were calculated by the binomial distribution method. The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate 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

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

1.3 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.:

1 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.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

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.
Sub-lethal effects of exposure were observed in the 1.0 mg/L loading rate WAF test concentration. This response was reduced mobility.
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.
The 10 and 100 mg/L loading rates were observed to be dispersions, therefore microscopic observations were conducted on a representative number of immobilized daphnids. Microscopic observations showed that no undissolved test item was adhered to the daphnids.
Chemical analysis of the 10 and 100 mg/L loading rate test preparations at 0 hours (see Annex 4) showed measured test concentrations of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.025 mg/L and 2.4 mg/L respectively were obtained. 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 the freshly prepared test preparations at 0 and 24 hours (see Annex 4) showed measured test concentrations to range from less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.025 mg/L, to 1.4 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from less than the LOQ to 0.99 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 using the binomial distribution method at 24 and 48 hours based on the nominal loading rates gave the following results:

Time (h) EL50 (mg/L Loading Rate WAF) 95% Confidence limits(mg/L Loading Rate WAF)
24 4.2 3.2 – 5.6*
48 1.3 1.0 – 1.8*

* Loading rates resulting in 0% and 100% immobilization respectively.

The No Observed Effect Loading rates after 24 and 48 hours exposure were 3.2 and 1.0 mg/L loading rate WAF respectively. Correspondingly the Lowest Effect Loading rates were considered to be 5.6 and 1.8 mg/L loading rate WAF.

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 22”C 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 754 to 812 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 the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear colorless water columns with globules of test item. After 23 hours stirring and a 1-Hour standing period the 1.0, 1.8, 3.2 and 5.6 mg/L loading rates were observed to remain as at the start of stirring whilst the 10 mg/L loading rate was observed to be a slightly cloudy water column with test item on the surface. Examination of the 10 mg/L loading rate showed the water column to be slightly cloudy and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). As a precautionary measure, all loading rates were filtered through a glass wool plug.

Results with reference substance (positive control):

A positive control (Envigo Study Number XT01HS) 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, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the 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 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) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 1.3 1.0-1.8 1.0 1.8
48 1.2 1.1-1.3 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

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	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	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	5	5	5	5	20	100	A/I	A/I	A/I	A/I
10	5	5	5	5	20	100	A/I	A/I	A/I	A/I

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	5	5	5	5	20	100	A/I	A/I	A/I	A/I
3.2	5	5	5	5	20	100	A/I	A/I	A/I	A/I
5.6	5	5	5	5	20	100	A/I	A/I	A/I	A/I
10	5	5	5	5	20	100	A/I	A/I	A/I	A/I

R₁– R₄= Replicates 1 to 4

N = Normal

A/I = All daphnia immobilized

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) No Observed Effect Loading Rate (NOEL) (mg/L) Lowest Observed Effect Loading Rate (LOEL) (mg/L)
48 1.3 1.0 - 1.8 1.0 1.8

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, "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, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of 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 freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.025 mg/L, to 1.4 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to range from less than the LOQ to 0.99 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 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	1.3	1.0 - 1.8	1.0	1.8

Description of key information

Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:

Time Point (h)	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	1.3	1.0 - 1.8	1.0	1.8

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.3 mg/L