Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

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:

11 December 2012-19 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate aliquots (ca. 20 mL) were removed from each tank at the commencement of the definitive test as 0 h samples and at test termination as 96 h samples.

Test samples were analysed according to the procedure established and validated under Charles River Study No. 220937, Procedure No. AP.2093.01.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test solutions were prepared by addition of weighed amounts of the substance coating additives (at nominal concentrations of 62.62 mg, 125.05 mg, 250.13 mg, 500.00 mg and 1000.12 mg for tanks at 3.125, 6.25, 12.5, 25 and 50 mg/L, respectively) to 20 L of RFW and ultrasonicating for ca. 10-15 min to aid dissolution. The control tank contained RFW only. One tank was prepared at each test concentration. Seven rainbow trout were added to each tank within 30 min of preparation.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Source: Brow Well Fisheries,Skipton, England
- Age at study initiation: 12 months

ACCLIMATION
- Acclimation period: 17 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: INICIO 917, fish fed at a rate of ca 1-2% of body weight per fish per day
- Feeding frequency: daily
- Health during acclimation (any mortality observed): all fish were in good health

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

The fish in the test were observed after 1, 4, 24, 48, 72 and 96 h. At the termination of the range finding and definitive tests, the length (from the tip of the snout to the caudal peduncle) and weight of each surviving fish was recorded.

Hardness:

80.0 mg CaCO3/L

Test temperature:

All test tank temperatures were in the range: 15.0-15.6°C

pH:

All test tank pH values were in the range: pH 7.24-8.07

Dissolved oxygen:

The dissolved oxygen contration for all test tanks were in the range:83.6-98.8%

Nominal and measured concentrations:

Nominal concentrations: 0, 3.125, 6.25, 12.5, 25 and 50 mg/L
Mean measured concentrations: 0, 3.00, 5.94, 12.1, 23.9 and 48.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: tanks of ca.25 L capacity (moulded glass construction and covered with Perspex lids to minimise dust contamination, evaporation loss and fish escaping)
- Type: open
- Fill volume: 20 L
- Aeration: throughout test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Conductivity of test tanks: 182.1-225µS/cm

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconsituted fresh water (prepared on site at Charles River)
- Intervals of water quality measurement: measured at test initiation and at 24 hour intervals in all test tanks

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0, 0.1, 1, 10 and 100 mg
- Results used to determine the conditions for the definitive study:
At 1 h exposure, all three fish at 100 mg/L were lying on the base of the tank in an inverted position, showing only limited movement. These fish were therefore removed and humanely euthanized to avoid additional stress. No further mortality or adverse effects were noted in any surviving fish throughout the 96 h test period.
The test solution at 100 mg/L appeared very slightly opaque. All other test solutions appeared clear and colourless throughout the test period. No precipitation of test item was observed.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

12.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: 0

Sublethal observations / clinical signs:

Measured Concentrations of the substance Coating Additives in Test Solutions During the Test

Nominal Concentration of the substance coating additives (mg/L)	Time (h)	Mean Measured Concentration of the substance coating additives (mg/L)	Overall Mean Measured Concentration of the substance coating additives (mg/L)	Deviation of 96 h Measured Concentration from 0 h Measured Concentration (±%)
Control	0 96	ND ND	ND ND	NA
3.125	0 96	3.01 2.99	3.00	-0.7
6.25	0 96	5.90 5.97	5.94	+1.2
12.5	0 96	12.0 12.1	12.1	+0.8
25	0 96	24.0 23.8	23.9	-0.8
50	0 96	48.6 48.1	48.4	-1.0

ND = Not detected

NA = Not applicable

Cumulative Mortality Recorded During the Test

Time (h)	Mean Measured Concentration of the substance coating additives (mg/L)
	Control	3.00	5.94	12.1	23.9	48.4
1	0	0	0	0	0	0
4	0	0	0	0	0	0
24	0	0	0	0	7	7
48	0	0	0	0	7	7
72	0	0	0	0	7	7
96	0	0	0	0	7	7

Seven fish were tested at each concentration.

Surviving Fish Lengths and Weights During the Test

Mean Measured oncentration of the substance coating additives (mg/L)	Fish No.	Length (cm)	Weight (g)
Control	1 2 3 4 5 6 7	5.56 4.67 5.70 4.78 5.38 5.98 5.53	2.4511 1.4784 2.5590 1.5285 2.3828 3.0801 2.3977
3.00	1 2 3 4 5 6 7	5.69 5.92 5.88 5.43 5.55 5.49 4.54	2.4876 2.6038 2.6342 2.2131 2.5492 2.1428 1.2804
5.94	1 2 3 4 5 6 7	5.99 5.52 5.37 5.79 5.31 4.73 5.55	3.2106 2.2365 2.2396 2.4599 2.0037 1.3484 2.5417
12.1	1 2 3 4 5 6 7	5.99 5.48 5.77 6.00 5.61 5.29 5.30	3.2104 2.3246 2.3196 3.2265 2.5034 2.0848 2.1136

Note: No fish survived at exposure concentrations of 23.9 and 48.4 mg/L.

Test Solution Quality Parameters During the Test

Time/Parameter	Mean Measured Concentration of the substance coating additives (mg/L)
	Control	3.00	5.94	12.1	23.9	48.4
0 hour   pH Temperature (°C) Conductivity (µS/cm) Dissolved Oxygen (% ASV)	7.43 15.1 183.9 87.7	7.42 15.2 183.0 83.6	7.49 15.4 182.8 85.7	7.51 15.4 182.5 84.2	7.49 15.4 182.1 86.0	7.43 15.4 183.0 86.6
24 hour   pH Temperature (°C) Conductivity (µS/cm) Dissolved Oxygen (% ASV)	7.47 15.4 220 89.9	7.32 15.5 220 90.1	7.27 15.6 221 90.4	7.24 15.2 222 92.3	7.26 15.2 223 94.4	7.27 15.4 225 95.1
48 hour   pH Temperature (°C) Conductivity (µS/cm) Dissolved Oxygen (% ASV)	8.04 15.4 213 97.6	8.07 15.4 215 96.9	7.93 15.4 216 92.9	7.88 15.0 216 96.5	7.89 15.1 215 98.3	7.92 15.3 217 98.8
72 hour   pH Temperature (°C) Conductivity (µS/cm) Dissolved Oxygen (% ASV)	7.64 15.4 219 93.0	7.41 15.4 219 91.1	7.24 15.4 220 90.0	7.25 15.0 221 93.3	7.28 15.1 219 95.0	7.24 15.3 220 96.6
96 hour   pH Temperature (°C) Conductivity (µS/cm) Dissolved Oxygen (% ASV)	7.88 15.4 208 90.1	7.83 15.4 207 88.4	7.79 15.4 207 89.4	7.83 15.1 209 87.2	7.85 15.1 208 88.4	7.86 15.3 208 90.9

ASV = Air saturation value

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 96 h LC50 and NOEC of the test substance for rainbow trout was estimated to be 18.0 and 12.1 mg/L respectively, based on arithmetic mean measured concentrations

Executive summary:

A study was conducted to determine the acute toxicity of test substance to rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 203, in compliance with GLP. Based on the results of a range finding test, the definitive test was conducted at nominal concentrations of 0, 3.125, 6.25, 12.5, 25 and 50 mg/L under static conditions. One tank was prepared at each test concentration and 7 fish added to each. The total test period was 96 h and samples for analytical confirmation of actual exposure concentrations were taken at the start (0 h) and at the end of the test (96 h). Chemical analysis of test samples taken at 0 and 96 h indicated that concentrations of the test substance were maintained within the range 99.0-101.2% of the initial measured values. No undissolved test substance was observed in the test tanks throughout the test period. Based on these results, the mean measured concentrations of the test substance were calculated to be 0, 3.00, 5.94, 12.1, 23.9 and 48.4 mg/L, respectively. The fish were observed at regular intervals for mortality and sub-lethal effects (length and body weight) in the surviving fish. The test conditions (pH, temperature, conductivity and dissolved oxygen) were measured daily during the test and found to be within the standard ranges. No fish survived in the top two test concentrations. Due to the lack of fractional mortality data at the 24, 48, 72 and 96 h time points, LC50 values were estimated by taking the arithmetic mean of the 0% and 100% mortality concentrations. The no observed effect concentration (NOEC) was estimated for mortality.  Under the study conditions, the 96 h LC50 and NOEC of the test substance for rainbow trout was estimated to be 18.0 and 12.1 mg/L respectively, based on arithmetic mean measured concentrations (Knight, 2013).

Description of key information

The 96-h LC50, NOEC of the test substance in fish was determined to be 18 mg/L and 12.1 mg/L (measured; arithmetic mean) respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

18 mg/L

Additional information

A study was conducted to determine the acute toxicity of test substance to rainbow trout (Oncorhynchus mykiss) according to OECD Guideline 203, in compliance with GLP. Based on the results of a range finding test, the definitive test was conducted at nominal concentrations of 0, 3.125, 6.25, 12.5, 25 and 50 mg/L under static conditions. One tank was prepared at each test concentration and 7 fish added to each. The total test period was 96 h and samples for analytical confirmation of actual exposure concentrations were taken at the start (0 h) and at the end of the test (96 h). Chemical analysis of test samples taken at 0 and 96 h indicated that concentrations of the test substance were maintained within the range 99.0-101.2% of the initial measured values. No undissolved test substance was observed in the test tanks throughout the test period. Based on these results, the mean measured concentrations of the test substance were calculated to be 0, 3.00, 5.94, 12.1, 23.9 and 48.4 mg/L, respectively. The fish were observed at regular intervals for mortality and sub-lethal effects (length and body weight) in the surviving fish. The test conditions (pH, temperature, conductivity and dissolved oxygen) were measured daily during the test and found to be within the standard ranges. No fish survived in the top two test concentrations. Due to the lack of fractional mortality data at the 24, 48, 72 and 96 h time points, LC50 values were estimated by taking the arithmetic mean of the 0% and 100% mortality concentrations. The no observed effect concentration (NOEC) was estimated for mortality.  Under the study conditions, the 96 h LC50 and NOEC of the test substance for rainbow trout was estimated to be 18.0 and 12.1 mg/L respectively, based on arithmetic mean measured concentrations (Knight, 2013).