Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-(2-aminomethylethyl)-.omega.-(2-aminomethylethoxy)-

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-07-28 to 2005-11-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

No information given on test substance purity or stability (responsibility of the Sponsor). No analytical monitoring of the test concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Saltwater species used, no information on purity or stability of test substance.

GLP compliance:

yes

Specific details on test material used for the study:

- Name as cited in study report: Jeffamine D-230
- Color: colorless
- Lot/Batch number: 4W512
- Data receipt 2005-07-05 727.9 g; 2005-07-13 750.6 g

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dissolution of test substance directly into dilution water
- Controls: Plain dilution water control and positive control with 32 mg/L sodium dodecyl sulfate

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Source: STILLMEADOW laboratory in-house culture.
- Age at study initiation (mean and range, SD): 14 days.
- Length at study initiation (length definition, mean, range and SD): The longest fish was not more than twice the length of the shortest.
- Weight at study initiation (mean and range, SD): < 3 g
- Method of breeding: in-house culture.
- Feeding during test: Not fed during test.
- Frequency: Fed daily until 48 h prior to test initiation.

ACCLIMATION
- Acclimation period: At least 7 days.
- Acclimation conditions (same as test or not): Same as test.
- Type and amount of food: Not specified.
- Feeding frequency: Daily

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Test temperature:

At 6 h- 20 °C
At 96 h- 21 °C

pH:

Range at test initiation-8.3-10.1
Range at test conclusion-8.0-9.3

Dissolved oxygen:

Range at test initiation-6.8-7.2 mg/L
Range at test conclusion-5.6-6.0 mg/L

Salinity:

Range at test initiation-21-24 ppt
Range at test conclusion-23-24 ppt

Nominal and measured concentrations:

Nominal concentrations (Definitive test): 0, 500, 600, 720, 864, 1037 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers, held in a environmentally controlled chamber.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, 1 L
- Aeration: none
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Biomass loading rate: 0.8 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic seawater
- Culture medium different from test medium: no
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 h light/ 8 h dark


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality; 0, 6, 24, 48, 72 and 96 h. Test containers observed daily for physical abnormalities, curved spine, sluggishness.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:~1.2
- Range finding study: Yes
- Test concentrations: 1, 10, 50, 100, 500 and 1000 mg/L (for range-finding)
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Sodium dodecyl sulfate (32 mg/L, JT Baker Lot # x27580)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

772.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL 728.25-818.68 mg/L

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

600 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Some fish sluggish at 864 mg/L and higher at 6 h; erratic swimming at highest concentration at 6 h. No physical abnormalities recorded.
- Mortality of control: None
- Other adverse effects control: None

Results with reference substance (positive control):

- Results with reference substance valid? Validity criteria not reported.
- Mortality: 100% at 6 h.

Reported statistics and error estimates:

The control was used to compare the mortality endpoints of the test concentration. The LC50 was determined using the Trimmed Spearman-Karber linear regression model.

Sublethal observations / clinical signs:

Table 1: Definitive Test Results

Conc. (m/L)	Total Number of Surviving Organisms
	0 h	6 h	24 h	48 h	72 h	96 h
0	30	30	30	30	30	30
500	30	27	27	27	26	26
600	30	29	29	29	28	28
720	30	26	25	18	18	17
864	30	26	14	11	11	11
1037	30	22	8	1	1	1
C2 SDS	30	0	0	0	0	0

 

Validity criteria fulfilled:

yes

Remarks:

Control mortality < 10%

Conclusions:

The 96 hr LC50 for the test subsqtance to sheepshead minnows was found to be 772.14 mg/L, with 95% confidence limits of 728.25-818.68 mg/L. The 96 hr NOEC was determined to be 600 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-10-25 to 2010-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name as cited in study report: POPDA
- Color: clear, colorless
- Lot/Batch number: LL100365
-Purity: 99.7%
- Data receipt: 2010-09-21
- Exiry date: 2011-12-31
- Storage: in the dark

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the 15 mg/l test group at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. The 0 and 24-Hour samples were stored at approximately -20ºC prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Method:
For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap water.
An amount of test item (1000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 500 ml to give a 2000 mg/l stock solution. An aliquot (300 ml) of this solvent stock solution was diluted in a final volume of 40 litres of dechlorinated tap water to give the 15 mg/l test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Controls:
Maintained under identical conditions but not exposed to to test material.

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 4 August 2010. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 13 October 2010 to 25 October 2010. 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 approximately 14ºC with a dissolved oxygen content of greater than or equal to 9.5 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 zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 7.0 cm (sd = 0.5) and a mean weight of 4.16 g (sd = 1.01) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.73 g bodyweight/litre.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test Water
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in attached Appendix 2.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 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.

Hardness:

Total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The test vessels were sealed and maintained at approximately 14ºC

pH:

The pH was measured using a Hach hq30d pH and dissolved oxygen meter.
pH range of 7.4-7.8.
Please see Physico-Chemical Measurements appendix 4(attached background material).
EXAMPLE

Dissolved oxygen:

The dissolved oxygen concentration was measured using a Hach hq30d pH and dissolved oxygen meter.
Please see Physico-Chemical Measurements appendix 4 (attached background material) for dissolved oxygen results.

Salinity:

Not applicable

Nominal and measured concentrations:

The test was conducted according to the threshold approach at a single concentration equivalent to the lowest EC50 value obtained from the Acute Toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number: 41003967) and the Algal Growth Inhibition test (Harlan Laboratories Ltd Project Number: 41003964). The results from the Acute Toxicity to Daphnia magna test gave an EC50 of 80 mg/l. The ErC50 for the Algal Growth Inhibition test was 15 mg/l.
Based on these results the test was conducted at a single test concentration of 15 mg/l to determine if the LC50 was higher than this lowest EC50 and hence that fish were not the most sensitive species.

Details on test conditions:

TEST SYSTEM
Test vessel:
In the test 40 litre glass exposure vessels were used for each 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 approximately 14ºC 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 ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.
Any mortalities and sub-lethal effects of exposure were recorded at 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.

TEST MEDIUM / WATER PARAMETERS
Source/preparation of dilution water:
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3.

OTHER TEST CONDITIONS
The test vessels were then sealed and maintained at approximately 14ºC 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.

Physico-chemical measurements
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 hq30d pH and oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sub-lethal effects of exposure were recorded at 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.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Not stated

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Not stated

Details on results:

Definitive Test
Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 1.
There were no mortalities in 14 fish exposed to a test concentration of 15 mg/l for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LC50 (mg/l)
3 >15
6 >15
24 >15
48 >15
72 >15
96 >15
The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 15 mg/l and the No Observed Effect Concentration (NOEC) to be 15 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

Sub-lethal effects
There were no sub-lethal effects of exposure observed in 14 fish exposed to a test concentration of 15 mg/l for a period of 96 hours.

Observations on test item solubility
The test preparation was observed to be a clear, colourless solution throughout the duration of the test.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4. Temperature was maintained at approximately 14ºC throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the freshly prepared media for the control and 15 mg/l test concentration throughout the test.
The oxygen concentration in some of the test vessels at was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Verification of test concentrations
Analysis of the test preparations at 0, 24 and 96 hours (see attached Appendix 3) showed measured test concentrations to range from 96% to 102% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

An estimate of the LC50 values was given by inspection of the mortality data.

Sublethal observations / clinical signs:

Table 1              Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (mg/l)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
15	0	0	0	0	0	0	0

  Appendix 4      Physico-Chemical Measurements

Nominal Concentration (mg/l)	Time (Hours)
	0 Hours (Fresh Media)				24 Hours (Old Media)				24 Hours (Fresh Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	7.9	10.7	102	13	8.2	9.9	96	14	7.9	10.7	102	13
15	8.3	10.6	101	13	8.3	10.0	97	14	8.2	10.7	104	14

 

Nominal Concentration (mg/l)	Time (Hours)
	48 Hours (Old Media)				48 Hours (Fresh Media)				72 Hours (Old Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	8.2	10.0	97	14	7.9	10.8	105	14	8.2	9.9	98	14
15	8.3	9.8	95	14	8.3	10.7	104	14	8.3	9.7	94	14

 

Nominal Concentration (mg/l)	Time (Hours)
	72 Hours (Fresh Media)				96 Hours (Old Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C
Control	7.6	10.7	104	14	8.3	9.9	96	14
15	8.2	10.6	103	14	8.3	9.8	95	14

 

*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 15 mg/l. Correspondingly the No Observed Effect Concentration was 15 mg/l.
This test was therefore considered valid based on the threshold effect as it showed an LC50 greater than the lowest EC50 from the algae and daphnia studies indicating that fish were not the most sensitive species of three trophic species tested.

Executive summary:

SUMMARY

Introduction.

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed that described in 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.

The test was conducted according to the threshold approach at a single concentration equivalent to the lowest EC₅₀value obtained from the Acute Toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number: 41003967) and the Algal Growth Inhibition test (Harlan Laboratories Ltd Project Number: 41003964). The results from the Acute Toxicity to Daphnia magna test gave an EC₅₀of 80 mg/l. The E_rC₅₀for the Algal Growth Inhibition test was 15 mg/l. Based on these results, fish were exposed, in a single group of seven, to an aqueous solution of the test item, at a single concentration of 15 mg/l for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC₅₀based on nominal test concentrations was greater than 15 mg/l and correspondingly the No Observed Effect Concentration was 15 mg/l.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 96% to 102% of nominal and so the results are based on nominal test concentrations only.

 

Description of key information

The 96 -h LC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to Rainbow  trout (Oncorhyncus mykiss) was found to be >15 mg/L. The NOEC was 15 mg/L (Goodband & Mullee, 2010). The 96 -h LC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to Sheepshead minnows (Cyprinodon variegatus) was found to be 772.14 mg/L, with 95% CL 728-818 mg/L (Stillmeadow Inc., 2005).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

15 mg/L

Marine water fish

Marine water fish

Effect concentration:

772.14 mg/L

Additional information

A static, 96-h acute limit test, conducted according to GLP and OECD Guideline 203, was run using freshwater Rainbow trout (Oncorhyncus mykiss) at a test concentration of 15 mg/L. Fish were not fed during the test. The 96 hr LC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to Rainbow trout was found to be >15 mg/L. The NOEC was 15 mg/L. Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 96% to 102% of nominal, so the results are based on nominal test concentrations only.

 

Another 96 -h static acute study, also by GLP and OECD 203, was conducted using the saltwater Sheepshead minnow (Cyprinodon variegatus). The fish used for the test were 14 day old fry from an in-house culture and weighed <3 g each. 3 replicates of 10 fish each were exposed per concentration and Control. Salinity of the synthetic seawater was 21 -24 ppt, and the temperature was 21 deg C. The concentrations were Control, 500, 600, 720, 864, 1037 mg/L. Fish were not fed during the test. The 96 hr LC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to Sheepshead minnows (Cyprinodon variegatus) was found to be 772 mg/L, with 95% CL 728-818 mg/L.