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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 23 July 2017 and 02 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study was conducted according to OECD TG 203, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 triethylenetetramine 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:

Total Organic Carbon (TOC) analysis was performed on the test solutions at 0, 24, 72 and 96 hours

Vehicle:

no

Details on test solutions:

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.

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 fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (22, 220 and 2200 mg) were each separately added to the surface of 22 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. Microscopic 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 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations were performed on the WAFs after filtering and showed that micro dispersions of test item were present, therefore Postlip filter paper was used in an attempt to remove as much undissolved test item as possible.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 24 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
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.

Experimental Preparation
Nominal amounts of test item (22, 39.6, 70.4, 123.2 and 220 mg) were each separately added to the surface of 22 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. Visual 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 75-100 mL discarded). Postlip filter paper was also used for the 0, 24 and 48 hour WAF preparations as per the range-finding test. Visual observations of the WAFs were performed after filtering and showed all test preparations to be cloudy dispersions, the turbidity of which increased with increased loading rate.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 12 September 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 02 October 2017 to 09 October 2017. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at 14”C to 15”C with a dissolved oxygen content of greater than or equal to 9.6 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm (sd = 0.2) and a mean weight of 0.88 g (sd = 0.15) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.31 g bodyweight/liter.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

14 - 16°C

pH:

7.1 - 8.1

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.
Chemical analysis of the 1.0 and 10 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of 0.44 and 2.1 mg/L respectively were obtained. A decline in measured test concentrations was observed at 24 hours to 0.29 and 1.4 mg/L indicating that the test item was unstable under the conditions of the test.

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 Water Accommodated Fraction (WAF) of the test item.

Validation of Mixing Period
Preliminary work (see Annex 2) 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 fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (22, 220 and 2200 mg) were each separately added to the surface of 22 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. Microscopic 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 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations were performed on the WAFs after filtering and showed that micro dispersions of test item were present, therefore Postlip filter paper was used in an attempt to remove as much undissolved test item as possible.
In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at approximately 15 C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions. Each 25-30 liter test and control vessel contained 20 liters of test media and was covered to reduce evaporation. After 1, 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.
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 24 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
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.

Experimental Preparation
Nominal amounts of test item (22, 39.6, 70.4, 123.2 and 220 mg) were each separately added to the surface of 22 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. Visual 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 75-100 mL discarded). Postlip filter paper was also used for the 0, 24 and 48 hour WAF preparations as per the range-finding test. Visual observations of the WAFs were performed after filtering and showed all test preparations to be cloudy dispersions, the turbidity of which increased with increased loading rate.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours (see Annex 3).

Exposure Conditions
As in the range-finding test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 15”C to 16”C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

ASSESSMENTS

Test Organism Observations
Any mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 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.

Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period.

Chemical Analysis of Test Loading Rates
Water samples were taken from the control and all surviving test groups at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described

Data Evalaution
Statistical Analysis
The LL50 values and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The LOEL and the NOEL at 3, 6, 24, 48, 72 and 96 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).

Data Validation
The results of the test are considered valid if the following criteria are met:
In the control, not more than one of the fish should die or show signs of stress during the 96 hours.
The dissolved oxygen concentration at the end of the test should be ≥60% of Air Saturation Values (ASV) in the control and test vessels.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Details on results:

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.

Range-finding Test
The results showed no mortalities at 1.0 mg/L loading rate WAF. However, mortalities were observed at 10 and 100 mg/L loading rate WAF.
After approximately 3 hours exposure 3 out of 3 fish at 10 mg/L loading rate WAF and after approximately 25 minutes exposure 3 out of 3 fish at 100 mg/L were observed to be moribund. These fish were all observed to be dead at the following observational time point
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 showed measured test concentrations of 0.44 and 2.1 mg/L respectively were obtained (see Annex 3). A decline in measured test concentrations was observed at 24 hours to 0.29 and 1.4 mg/L indicating that the test item was unstable under the conditions of the test.

Definitive Test
Chemical Analysis of Test Loading Rates
Analysis of the freshly prepared media at 0 and 72 hours showed that measured concentrations of between 0.11 and 3.5 mg/L were obtained. Analysis of the old or expired test preparations at 24 and 96 hours showed that measured concentrations of between 0.086 and 3.2 mg/L were obtained.
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.

Mortality Data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2
Analysis of the mortality data by Probit analysis using Linear Maximum-Likelihood regression at 3, 6, 24, 48, 72 and 96 hours based on the nominal test concentrations gave the following results:

Time (h) LL50 (mg/L Loading Rate WAF) 95% Confidence limits(mg/L Loading Rate WAF)
3 6.9 5.9 – 8.0
6 4.2 3.6 – 4.9
24 2.9 Not Determined
48 1.9 Not Determined
72 1.8 Not Determined
96 1.8 Not Determined

The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 1.0 mg/L, the lowest loading rate resulting in 100% mortality to be 3.2 mg/L. The Lowest Observed Effect Loading rate (LOEL) was considered to be 1.8 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) to be 1.0 mg/L loading rate WAF.


Sub-Lethal Effects
Sub-lethal effects of exposure were observed at the 1.0 mg/L loading rate WAF and above. These responses were swimming at the bottom of the tank, sat at the bottom of the tank, moribund and increased pigmentation. (Table 3)
After approximately 45 hours exposure 1 out of 7 fish at 1.8 mg/L loading rate WAF were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) these fish were killed and classed as mortalities for the following observational time point.

Validation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.

Water Quality Criteria
Water temperature was maintained at 15”C to 16”C 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 each 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 at 0, 24, and 48 hours the 1.0, 1.8, 3.2, 5.6, and 10 mg/L loading rates were observed to be clear colourless water columns with oily globules of test item floating at the surface of the water. At the start of the mixing period at 72 hours, the 1.0 and 1.8 mg/L loading rates were observed to be clear colourless water columns with test item floating at the surface of the water. After 23 hours stirring and a 1-Hour standing period the 1.0, 1.8, 3.2, 5.6, and 10 mg/L loading rates at 0, 24 and 48 hours were observed to be cloudy dispersions increasing in turbidity with increasing concentrations. At 72 hours the 1.0 and 1.8 mg/L loading rates were observed to be slightly cloudy homogenous dispersions. Visual examination of the WAFs at 48 and 72 hours showed cloudy dispersions. No visual or microscopic observations were made during siphoning, however visual observations made at the end of the settlement period show that the WAF’s were observed to be cloudy dispersions and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). Postlip filter paper was also used as per the range-finding test. Visual examination after filtering showed the glass wool plug and filter paper had not removed the dispersed material and the solutions remained cloudy. During the test the 1.0 and 1.8 mg/L loading rates were observed to be slightly cloudy homogenous dispersions and the 3.2, 5.6 and 10 mg/L loading rates were observed to be cloudy homogenous dispersions.

Sublethal observations / clinical signs:

Table 2       Cumulative Mortality Data in the Definitive Test 

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 7)							% Mortality
	1  Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	2	3	4	57
3.2	0	0	0	6	7	7	7	100
5.6	0	1	7	7	7	7	7	100
10	1	7	7	7	7	7	7	100

 

Table 4            Sub-lethal Effects of Exposure in the Definitive Test

Nominal Loading Rate (mg/L)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	7	7	7	7	7	7	7
1.0	No abnormalities detected	7	7	7	7	7	1	7
1.0	Swimming at the bottom of the tank	0	0	0	0	0	6	0
1.8	No abnormalities detected	7	7	7	3	0	1	3
1.8	Swimming near the bottom of the tank	0	0	0	4	0	0	0
1.8	Moribund	0	0	0	0	1	0	0
1.8	Sat on the bottom of the tank with increased pigmentation	0	0	0	0	5	0	0
1.8	Sat on the bottom of the tank	0	0	0	0	0	3	0
3.2	No abnormalities detected	7	7	7	0	0	0	0
3.2	Sat on the bottom of the tank with increased pigmentation	0	0	0	1	0	0	0
5.6	No abnormalities detected	7	0	0	0	0	0	0
5.6	Swimming at the bottom of the tank	0	6	0	0	0	0	0
10	Swimming at the bottom of the tank	6	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.

Conclusions:

Exposure of the freshwater fish rainbow trout (Oncorhynchus mykiss) to the test item has been investigated and gave the following results:
Time Point (Hours) LL50 (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)
96 1.8 Not Determined 1.0 1.8

Executive summary:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish Acute Toxicity Test" referenced as Method C.1 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, fish were exposed, in groups of seven, 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 a period of 96 hours at a temperature of 15 to 16 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 0.11 to 3.5 mg/L. Chemical analysis of the aged test preparations at 24 and 96 hours showed measured test concentrations to range from 0.086 to 3.2 mg/L.

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 rainbow trout to the test item gave the following results:

Time Point (Hours)	LL50 (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)
96	1.8		Not Determined		1.0	1.8

Description of key information

Exposure of the freshwater fish rainbow trout (Oncorhynchus mykiss) to the test item has been investigated and gave the following results:

Time Point (h)	LL50 (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)
96	1.8	Not determined	1.0	1.8

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.8 mg/L