2-(2,6-diethyl-4-methyl-phenyl)propanediamide

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:

02 November 2003 to 07 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for analysis were taken from the centre of the test solutions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution was prepared by adding a known amount of the test material directly to 30 litres of dilution water in the test vessel. The test solutions was stirred for 16.5 hours prior to the addition of the test organisms. The solution was yellow in colour.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Length at study initiation (length definition, mean, range and SD): 49 to 57 mm (mean 54 mm)
- Weight at study initiation (mean and range, SD): 1.43 to 2.68 g (mean 2.12 g)
- Feeding during test: None

ACCLIMATION
- Acclimation period: A minimum of 7 days
- Acclimation conditions (same as test or not): 25 % seawater. Prior to the test, the fish were acclimatised to the test temperature (15 ± 1 °C)
- Feeding frequency: During the holding and acclimatisation periods, the fish were fed appropriate amounts of a commercial fish food. Food was withheld from the fish for at least 24 hours prior to the commencement of the test.
- Health during acclimation (any mortality observed): The last medication given to the fish was 170 mg/L formalin treatment given for 1 hour on the last day of the holding period. Less than 1 % mortality was observed in the batch of fish in the 7 days prior to the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

53.7 mg/L as CaCO3

Test temperature:

15 ± 1 °C

pH:

7.32 to 7.71

Dissolved oxygen:

9.49 to 9.96 mg/L
>94 % of the air saturation value

Nominal and measured concentrations:

100 mg/L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel material, size, headspace, fill volume: Single borosilicate glass vessels 460 mm x 305 mm x 310 mm. Maximum capacity of 43 L and a working volume of 30 L.
- Aeration: Gently aerated
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): triplicate
- Biomass loading rate: 0.71 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water, which had been passed through activated carbon coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. Salts were added to maintain minimum hardness levels and the treated water was then passed through an UV steriliser to a second set of 20 and 10 µm filters.
- Chlorine: <2 µg/L
- Alkalinity: 29.8 mg/L as CaCO3
- Ammonia: 1.0 µg/L as NH3-N
- Conductivity: 274 µS/cm at 25 °C
- Culture medium different from test medium: The fish were kept in 25 % sea water.
- Intervals of water quality measurement: The pH and conductivity of the laboratory dechlorinated water supply were monitored every week day. Hardness, alkalinity and free residual chlorine were measured once a week. Total ammonia was measured once a month. The dechlorinated water supply was also monitored periodically for chemical oxygen demand, total organic carbon, total suspended solids, a range of cations and anions, trace metals, pesticides and other hydrocarbons.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours fluorescent light and 8 hours dark with 20 minute dawn and dusk transition periods commencing at 06:00 and 21:40 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daily measurements of the test solutions were undertaken throughout the 96 hour period for pH and dissolved oxygen concentration using calibrated meters. Temperature values were determined daily using a mercury-in-glass thermometer calibrated to 0.1 °C and conforming to BS593. Hourly temperature measures were also recorded automatically in the dilution water control using an electronic recording system.

Observations for mortalities and symptoms of toxicity were made at 3, 24, 48, 72 and 96 hours.

Reference substance (positive control):

no

Duration:

3 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: The only symptom of toxicity observed in this study was sounding.
- Mortality of control: No organisms died in the controlce in test medium:

Sublethal observations / clinical signs:

Table 1: Analytical results

Nominal concentration of test material (mg/L)	Measured concentration of test material (mg/L)				Mean measured concentration over the test duration^a(mg/L)	Mean measured concentration as percentage of nominal concentration	Percentage loss in conc over test duration^a
	0 hours	% of nominal	96 hours	% of nominal
Dilution water control	<0.012	-	<0.012	-	<0.012	-	-
100	110^c	110	110^d	110	110	110	0

^aCalculated using the arithmetic mean of the 0 and 96 hour results.

^b100-[(final analysed concentration/concentration at 0 hours)x100

^cMean triplicate analyses.

^dMean triplicate analyses. 

Table 2: Symptoms of toxicity observed

Nominal concentration of test material (mg/L)	Symptoms observed
	3 h	24 h	48 h	72 h	96 h
100	A	A	A	Ca	A

A = 10% or less of test population dead or exhibiting symptoms of toxicity

B = between 11% and 30% of test population dead or exhibiting symptoms of toxicity

C = more than 30% of test population dead or exhibiting symptoms of toxicity

a - sounding 

Table 3: pH, Dissolved oxygen concentrations and temperature of test vessels

Nominal concentration of test material (mg/L)	0 hours				24 hours				48 hour				72 hours				96 hours
	DO (mg/L)	DO%^a	pH	Temp (°C)	DO (mg/L)	DO%	pH	Temp (°C)	DO (mg/L)	DO%	pH	Temp (°C)	DO (mg/L)	DO%	pH	Temp (°C)	DO (mg/L)	DO%	pH	Temp (°C)
Dilution water control	9.82	98	7.71	15	9.49	94	7.45	14.8	9.83	98	7.38	14.6	9.79	97	7.52	14.6	9.66	96	7.42	14.6
100	9.96	99	7.64	15	9.56	95	7.42	14.7	9.79	97	7.32	14.5	9.77	97	7.52	14.6	9.67	96	7.39	14.6

Table 4: Dilution water quality

Test day	pH	Conductivity at 25 °C (µS/cm)	Alkalinity as CaCO3 (mg/L)	Hardness total as CaCO3 (mg/L)	Chlorine (as Cl2)		Total ammonia as NH3 -N µg/L
					Free residual (µg/L)	Combined residual (µg/L)
-1	7.81	2.74	29.8	53.7	<2	<2	1

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, there were no mortalities observed during the test, the only symptom of toxicity observed was sounding. The 96 h LC50 of the test material could not be derived as no mortalities were observed during the test therefore the NOEC of the test material to Oncorhynchus mykiss was determined to be 100 mg/L. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The acute toxicity of the test material to fish was investigated in Oncorhynchus mykiss (rainbow trout) in accordance with the standardised guidelines OECD 203, EU Method C.1 and EPA OPPTS 850:1075. The fish were exposed to the test material in a limit test (100 mg/L) under static conditions. The mean measured concentration of the test substance was 110 % of the nominal exposure concentration; the results and conclusions were therefore based on the nominal concentrations. No mortalities were observed during the course of the study with the only symptoms of toxicity that were observed being sounding. The NOEC of the test material under the conditions of the test was therefore determined to be 100 mg/L.

Description of key information

LC50 (96 h): >100 mg/L, Oncorhynchus mykiss, OECD 203, Maynard 2003

LC50 (96 h): >107 mg/L, Gobiocypris rarus, OECD 203, Guixiang 2016

Additional information

The acute toxicity of the test material to fish was investigated in rainbow trout (Oncorhynchus mykiss) in accordance with the standardised guidelines OECD 203, EU Method C.1 and EPA OPPTS 850:1075. The fish were exposed to the test material in a limit test (100 mg/L) under static conditions. The mean measured concentration of the test material was 110% of the nominal exposure concentration; the results and conclusions were therefore based on the nominal concentrations. No mortalities were observed during the course of the study with the only symptoms of toxicity that were observed being sounding. The NOEC of the test material under the conditions of the test was therefore determined to be 100 mg/L and the LC50 >100 mg/L.

The low acute toxicity to fish was confirmed in a second GLP study on rare minnow (Gobiocypris rarus) according to OECD TG 203, which found a 96-hour LC50 value >107 mg/L. This study was conducted as a limit test at a nominal test concentration of 100 mg/L, because no mortality had been seen in a preliminary study at 1, 10 or 100 mg/L. The mean concentration determined by UPLC over the test period of 96 hours was 107 mg/L.