N-(2-hydroxypropyl)oleamide

Long-term toxicity to fish

Administrative data

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 19 October 2016 and 13 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxypropyl)oleamide

EC Number:

203-828-2

EC Name:

N-(2-hydroxypropyl)oleamide

Cas Number:

111-05-7

Molecular formula:

C21H41NO2

IUPAC Name:

N-(2-hydroxypropyl)oleamide

Test material form:

other: waxy solid

Specific details on test material used for the study:

Identification: N-(2-hydroxypropyl) Oleamide
Physical state/Appearance: Beige waxy solid
Batch: 160602013073 w/o solvent
Purity: 100%
Expiry Date: 30 May 2018
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding test:
Samples of the fresh test preparations were taken for chemical analysis on Days 0 and 2 and of the expired test preparations on Days 2 and 5. All samples were stored frozen prior to analysis.

Continuous-flow dosing trial:
Samples of the test preparations were taken on Days 1 and 2 for chemical analysis.

Definitive test:
Water samples were taken from the control, solvent control and 0.75 mg/L test groups from the freshly prepared bulk test preparation on Days 0, 2, 6, 11, 14, 18, 25 and 32 and from the old or expired media on Days 1, 4, 7, 12, 15, 19, 26 and 33 (Replicates R1 to R4 pooled) for quantitative analysis. The samples were analyzed on the day of sampling with the exception of the samples taken on Days 11 and 12 which were not analyzed.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

yes

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed (ECETOC, 1996 and OECD, 2000). A solvent stock solution was prepared by dissolving 300 mg of test item in a final volume of 10 mL of dimethylformamide. An aliquot of this solvent stock solution was dispersed in test water with the aid of magnetic stirring for approximately 15 minutes to give a 0.75 mg/L test concentration.

Range-findng test:
A nominal amount of test item (60 mg) was dissolved in dimethylformamide and the volume adjusted to 2 mL to give a 30 mg/mL solvent stock solution. A series of dilutions was made from the 30 mg/L solvent stock solution to produce 0.30 and 3.0 mg/L solvent stock solution. Aliquots (100 µL) of the solvent stock solutions were dispersed in 4 liters of test water with the aid of magnetic stirring for approximately 15 minutes to give the 0.0075, 0.075 and 0.75 mg/L test concentrations.
The control group was maintained under identical conditions but not exposed to the test item or solvent.

Continuous-flow Dosing Trial:
A nominal amount of test item (375 mg) was dissolved in dimethylformamide and the volume adjusted to 25 mL to give a 15 mg/mL stock solution from which a series of dilutions was made to give further stock solutions of 1.5 and 0.15 mg/mL. The stock solutions were freshly prepared each day.
Each of the stock solutions was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test:
A nominal amount of test item (300 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 30 mg/mL solvent stock solution.
On Days 0 through to Day 11 an aliquot (100 µL) of this solvent stock solution was dispersed in 4 liters of test water with the aid of magnetic stirring for approximately 15 minutes to give the 0.75 mg/L test concentration.
On Day 12 through to the end of the test, an aliquot (500 µL) of the 30 mg/mL solvent stock solution was dispersed in 20 liters of test water with the aid of magnetic stirring for approximately 15 minutes to give the 0.75 mg/L test concentration.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.

Test organisms

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were bred at Envigo Research Limited on 5 October 2016 and maintained in dechlorinated tap water with an activated carbon and biological filtration system.
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the seven days preceding the start of the test, the water temperature was controlled at approximately 23 ºC to 25 ºC with a dissolved oxygen content of greater than or equal to 7.6 mg O2/L. The breeding stock fish were fed ZM 400 flake food daily and frozen brine shrimp.
Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 09 March 2017 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

33 d

Test conditions

Hardness:

128 to 138 mg/L as CaCO3 at the start of the test and from 124 to 140 mg/L as CaCO3 at termination of the test

Test temperature:

23.3 ºC to 25.4 ºC

pH:

7.1 to 8.6

Dissolved oxygen:

2.8 and 9.1 mg O2/L (equivalent to 34% and 110% ASV)

Nominal and measured concentrations:

Analysis of the fresh test preparations on Days 0, 2, 6, 14, 18, 25 and 32 showed measured test concentrations to range from 0.49 mg/L to 0.90 mg/L. A decline in measured test concentration of the aged test preparations on Days 1, 4, 7, 15, 19, 26 and 33 was observed to be less than the LOQ in all instances and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The geometric mean measured test concentration was determined to be 0.081 mg/L.
Information provided by the Sponsor suggested that a reaction between the test item and the test medium and/or adsorption on fish and/or feed may occur, resulting in a lack of detection in the aged test solutions through a possible modification of the molecular mass and to a lack of detection in the aged test preparations, therefore results are expressed as the average of the initial measured concentration. The average initial measured concentration was determined to be 0.67 mg/L.

Details on test conditions:

TEST SYSTEM
Range finding test:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing newly laid eggs to a series of nominal test concentrations of 0.0075, 0.075 and 0.75 mg/L for a period of 15 days.
The test vessels each contained approximately 400 mL of test preparation from Day 0 to 7, increasing to approximately 800 mL from Day 7 to 15. Two replicate vessels were used for each control and test concentration.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 15 days under semi-static test conditions. The test preparations were renewed on Days 2, 5, 7, 9 and 12.
The test vessels were aerated via narrow bore glass tubes from Day 8 onwards. The eggs and larvae were not individually identified.
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily.
The larvae were fed freshly hatched special grade brine shrimp nauplii from Day 7 to Day 11. On Day 12 onwards, the larvae were fed freshly hatched basic grade brine shrimp nauplii.

Continuous-flow dosing trial:
A dosing trial was conducted to determine the stability of the test item under continuous-flow test conditions.
Dynamic continuous flow test conditions were employed in the test. Dilution water was dosed at a flow rate of 120 mL per minute using Tacmina solenoid-driven metering pumps and the solvent stock solutions at a flow rate of 0.36 mL per hour using a Harvard Syringe Pump. The dilution water and solvent stock solutions were combined in separate mixing vessels and delivered to the test vessels using a two way splitter at 30 mL per minute to each replicate to give test concentrations of 0.015, 0.15 and 1.5 mg/L.

Definitive test:
Based on the results of a preliminary range-finding test, newly laid fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a solution of the test item at a nominal concentration of 0.75 mg/L for a period of 33 days under semi-static test conditions.
In the definitive test 1 liter glass vessels were used from Day 0 to Day 12 and from Day 12 to the end of the test 5 liter glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 6, 800 mL from Day 6 to Day 12 and 4000 mL from Day 12 through to the end of the test. Four replicate flasks were used for each control and test concentration.
A semi-static test regime was employed in the test involving a renewal of the test preparations daily throughout the test, with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs. The test solutions were renewed daily to ensure that the concentrations of the test item remained near nominal and to prevent the build-up of nitrogenous waste products.
Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ± 1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.
The test vessels were aerated via narrow bore glass tubes from Day 7 onwards. The eggs and larvae were not individually identified.
The larvae were fed freshly hatched special grade brine shrimp nauplii from Day 6 to Day 11 and freshly hatched basic grade brine shrimp nauplii from Day 12 through to the end of the test.

TEST MEDIUM / WATER PARAMETERS
The test water used for the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Elga AC1) 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.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test Organism Observations
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus.
At the end of the test the length and wet weight of each surviving fish was determined.
Water Quality Criteria
Dissolved oxygen concentrations and pH were recorded before and after each test media renewal. The water temperature and light intensity were recorded daily throughout the test. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration was measured using a Hach Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored approximately every hour in Control Replicate R1 using a Testo temperature logger.
The water hardness was measured in the bulk test preparation at the start and in each vessel on termination of the test and was determined using the methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.67 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Survival and Growth

Effect concentrations 2

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

0.081 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival and growth

Details on results:

Range-finding Test
The results showed there was no difference between the control and test concentrations of 0.0075, 0.075 and 0.75 mg/L in terms of hatching, survival and growth.
No sub-lethal effects were observed at the test concentrations of 0.0075 and 0.75 mg/L, however, sub-lethal effects were observed at 0.075 mg/L. These sub-lethal effects observed were small and a bent spine, however, these observations only affected a single fish and were therefore considered not to be significant.
Based on this information, a single test concentration of 0.75 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 0.75 mg/L, no adverse reactions to exposure were observed.
Chemical analysis of the 0.075 and 0.75 mg/L fresh test preparations on Days 0 and 2 showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.020 mg/L, and 0.60 mg/L. Measured test concentration of the corresponding aged test preparations on Days 2 and 5 showed measured test concentrations of less than the LOQ.

Continuous-flow Dosing Trial
Analysis of the continuous-flow test preparations on Days 0 and 2 of dosing indicated that stable measured concentrations could not be maintained, therefore, the definitive test was conducted following a semi-static test design with daily renewal of the test solutions.

Definitive Test
Number of Eggs Hatching
The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed.
The start of egg hatching was observed on Day 3 of the test and completion of hatching was observed on Day 6 of the test. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.

Statistical analysis of the hatching data was carried out for the pooled controls and the test group. There were no statistically significant differences (P ≥ 0.05), between the pooled controls and the test group and therefore the "No Observed Effect Concentration" (NOEC) based on the average initial measured concentration for the number of eggs hatching was 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L).

Post-hatch Survival
The number of dead larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed.
There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.

Statistical analysis of the post-hatch survival data was carried out for the pooled controls test group. There were no statistically significant differences (P ≥ 0.05), between the pooled controls and the test group and therefore the "No Observed Effect Concentration" (NOEC) based on the average initial measured concentration for post-hatch survival was 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L).

Length and Weight Data
Statistical analysis of the fish body length data was carried out for the solvent control and the test group. There were no statistically significant differences (P ≥ 0.05), between the solvent control and the test group and therefore the "No Observed Effect Concentration" (NOEC) based on the average initial measured concentration for body length was 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L).

Statistical analysis of the fish wet weight data was carried out for the solvent control and the test group. There were no statistically significant differences (P ≥ 0.05), between the solvent control and the test group and therefore the "No Observed Effect Concentration" (NOEC) based on the average initial measured concentration for wet weight was 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L).
Given this information and data assessment above it was considered that no effect on survival or growth attributable to the test item was observed.

Observations
There were no sub-lethal effects observed in the test.

Any other information on results incl. tables

Number of Eggs Hatching

	Expressed as the Average Initial Measured Concentration (mg/L)	Expressed as the Geometric Mean Measured Concentration (mg/L)
LC10(Hatching)	> 0.67	> 0.081
LC50(Hatching)	> 0.67	> 0.081

Post-hatch Survival

	Expressed as the Average Initial Measured Concentration (mg/L)	Expressed as the Geometric Mean Measured Concentration (mg/L)
LC10(Post-hatch Survival)	> 0.67	> 0.081
LC50(Post-hatch Survival)	> 0.67	> 0.081

Length and Weight Data

	Expressed as the Average Initial Measured Concentration (mg/L)	Expressed as the Geometric Mean Measured Concentration (mg/L)
EC10(Length)	> 0.67	> 0.081
EC50(Length)	> 0.67	> 0.081

	Expressed as the Average Initial Measured Concentration (mg/L)	Expressed as the Geometric Mean Measured Concentration (mg/L)
EC10(Weight)	> 0.67	> 0.081
EC50(Weight)	> 0.67	> 0.081

Validation Criteria

The following validity criteria were achieved during the test:

	Required	Achieved
1)     Dissolved oxygen	60% - 100% ASV	34 to 110% ASV
2)     Water temperature
Between test chambers or successive days	± 1.5^oC	± 1.6^oC
Range	25 ± 1.5^oC	23.3 - 25.9^oC
3)     Hatching success rate*	>70%	96%
4)     Post-hatch survival^*	>75%	97%

*In control vessels

 Observations on Test Item Solubility

After dosing all test concentrations were observed to be clear, colourless solutions by visual inspection.

Hatching and Post Hatch Survival in the Definitive Test are presented in the table below.

Average Initial Measured Concentration (mg/L)		Hatching Rate (%)	Mean Hatching Rate (%)	Survival Rate (%)	Mean Survival Rate (%)
Control	R1	100	96	100	97
	R2	95		95
	R3	95		100
	R4	95		95
Solvent Control	R1	90	94	94	97
	R2	95		95
	R3	95		100
	R4	95		100
0.67 (GMM = 0.081)	R1	90	93	100	96
	R2	80		100
	R3	100		85
	R4	100		100

R₁– R₄= Replicates 1 to 4

GMM = Geometric Mean Measured Concentration

Fish Length and Weight Data are displayed in the following tables.

Fish	ControlR1		Control R2		Control R3		Control R4
	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)
1	12.41	12.9	12.44	11.0	20.74	71.5	21.32	84.5
2	21.07	89.7	13.76	16.9	21.59	77.6	17.75	70.0
3	15.46	22.7	22.36	105.4	21.42	84.0	16.85	36.0
4	21.59	77.7	20.87	74.4	21.46	82.4	18.41	55.2
5	14.92	23.1	21.29	86.2	13.40	14.4	21.97	106.4
6	12.77	11.4	22.45	108.4	21.21	95.2	23.31	121.2
7	19.18	68.7	21.29	79.7	21.95	90.6	22.93	121.4
8	21.08	78.6	23.43	120.1	21.81	104.1	23.06	107.2
9	22.93	101.1	22.86	101.5	21.79	81.7	15.04	26.7
10	19.93	17.0	13.07	14.5	21.18	85.8	20.14	67.8
11	21.31	74.5	24.90	144.5	20.79	81.2	21.92	94.7
12	21.20	93.1	14.03	19.2	14.47	22.2	22.65	99.7
13	15.87	28.0	15.97	30.1	23.22	116.4	23.03	111.9
14	21.53	88.0	22.32	96.0	13.41	16.4	22.86	96.1
15	21.79	77.9	16.63	33.4	15.77	28.9	21.92	82.0
16	22.27	90.0	15.60	24.7	22.68	95.7	18.62	50.0
17	20.14	61.0	24.33	118.9	18.55	60.1	17.10	38.4
18	22.10	92.0	20.04	57.0	21.26	83.4	14.32	18.2
19	20.53	68.7	-	-	22.64	105.2	-	-
20	22.94	98.2	-	-	-	-	-	-
Average	19.25	63.7	19.31	69.0	20.02	73.5	20.18	77.1

Group Mean Length: 19.69 mm

Group Mean Wet Weight: 70.8 mg

R₁– R₄= Replicates 1 to 4

- = No measurement recorded due to fish mortality

Fish	Solvent ControlR1		Solvent ControlR2		Solvent ControlR3		Solvent ControlR4
	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)	Length (mm)	Wet Weight (mg)
1	21.63	89.7	12.93	12.3	13.47	15.5	21.55	77.0
2	21.60	89.9	20.69	84.9	23.48	111.5	23.53	109.3
3	22.61	99.6	16.36	27.6	25.44	142.7	21.80	80.3
4	21.38	87.6	20.36	78.8	24.21	129.7	22.56	109.7
5	20.25	72.1	23.15	109.9	22.58	94.6	22.18	97.6
6	23.29	107.0	23.03	107.0	21.47	80.9	20.00	69.7
7	16.69	33.6	21.18	81.0	19.75	65.5	22.95	105.2
8	21.63	95.6	23.37	122.6	14.43	20.2	22.40	101.2
9	23.97	106.8	17.06	41.9	20.89	83.6	16.55	33.5
10	22.54	96.1	22.36	95.4	22.86	112.0	20.63	83.2
11	22.58	101.1	22.86	114.2	21.76	87.6	22.17	93.1
12	22.17	109.5	22.92	98.7	20.50	75.0	20.91	85.9
13	21.72	97.5	23.46	108.4	22.98	114.9	16.90	33.1
14	24.14	115.1	22.19	103.9	21.95	99.4	20.43	71.3
15	23.26	98.6	19.53	67.7	12.94	15.1	23.04	106.0
16	15.65	30.3	23.07	111.5	21.74	99.9	22.14	88.9
17	15.94	31.6	17.07	36.6	14.96	24.0	21.41	90.7
18	-	-	21.02	79.1	19.41	61.5	16.61	32.7
19	-	-	-	-	24.95	140.1	22.59	96.6
20	-	-	-	-	-	-	-	-
Average	21.24	86.0	20.70	82.3	20.51	82.8	21.07	82.4

Group Mean Length: 20.88 mm

Group Mean Wet Weight: 83.4 mg

R₁– R₄= Replicates 1 to 4

- = No measurement recorded due to fish mortality

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The application of the test item to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Concentration based on the average initial measured concentration was 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L).
This study showed that there were no toxic effects at the highest attainable test concentration.

Executive summary:

 Introduction

A study was performed to assess the effects of the test item on freshly hatched larvae of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.

Methods

Information provided by the Sponsor indicated the water solubility of the test item was approximately 0.75 mg/L using a solvent spike method of preparation.

Based on the results of a preliminary range-finding test, newly laid fathead minnow eggs
(4 replicates of 20 eggs per group) were exposed to a solution of the test item at a nominal concentration of 0.75 mg/L for a period of 33 days (28 days post-hatch) at a temperature of 23 ºC to 26 ºC under semi-static test conditions. The test solutions were renewed daily throughout the test with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs. An initial solvent stock solution was prepared by dissolving 300mg of test item in a final volume of10mL of dimethylformamide to give a
30mg/mL solvent stock solution. On Days 0 to 11, an aliquot (100 µL) of this solvent stock solution was dispersed in4liters of test water with the aid of magnetic stirring for approximately15minutes to give a 0.75mg/L test concentration. On Days 12 through to the end of the test, an aliquot (500 µL) of this solvent stock solution was dispersed in 20 liters of test water with the aid of magnetic stirring for approximately15 minutes to give a 0.75mg/L test concentration. 

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.

Results

Analysis of the fresh test preparations on Days 0, 2, 6, 14, 18, 25 and 32 showed measured test concentrations to range from 0.49 mg/L to 0.90 mg/L. A decline in measured test concentration of the aged test preparations on Days 1, 4, 7, 15, 19, 26 and 33 was observed to less than the LOQ in all instances and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The geometric mean measured test concentration was determined to be 0.081 mg/L.

Information provided by the Sponsor suggested that a reaction between the test item and the test medium and/or adsorption on fish and/or feed may occur, resulting in a lack of detection in the aged test solutions through a possible modification of the molecular mass and to a lack of detection in the aged test preparations, therefore results are expressed as the average of the initial measured concentration. The average initial measured concentration was determined to be 0.67 mg/L.

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to a an average initial measured concentration of 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L). The No Observed Effect Concentration based on the average initial measured concentration was 0.67 mg/L (corresponding to a geometric mean measured concentration of 0.081 mg/L).

This study showed that there were no toxic effects at the highest attainable test concentration.