4-mesyl-2-nitrotoluene

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jun 2005 to 02 Aug 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from one test chamber of each treatment and control group two days prior to test initiation. Samples were collected from alternating replicate test chambers of each treatment and control group on days 0, 7, 14, 21 and 28.
- Sampling method: Samples were taken from centre of the test vessels.
- Sample storage conditions before analysis: Samples were analysed either directly after sampling or were stored frozen for up to one week.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Application solutions of nominal 100 mg/L prepared by dissolving 15 g of the test substance in 150 L of dilution water. Appropriate proportions of this solution were added each cycle by a proportional diluter system.
- Controls: blank control of dilution water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Houghton Springs Fish Farm, Winterborne Houghton, Blandford Forum, Dorset, DT11 0PD
- Age at study initiation: Juvenile
- Wet weight at study initiation (mean ± SD): 1.13 ± 0.10 g
- Method of breeding: Not reported

FEEDING DURING TEST
- Food type: Commercially available diet (Ecostart pellet)
- Amount: 4% last measured mean wet weight (based on weighings at day 0 and day 14, corrected for any mortalities)
- Frequency: daily, except for 48 hours before start of test and 24 hours before days 14 and 28 when fish were not fed.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

Control: 221.6 mg CaCO3/L
Highest test concentration: 223.4 mg CaCO3/L

Test temperature:

12.7 - 14.2 °C

pH:

7.74 - 8.33

Dissolved oxygen:

80 - 97 % of air saturation value

Nominal and measured concentrations:

See Table 1. Results are reported as mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 30 L filled with approx. 20 L aqueous media
- Aeration: Via glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): Proportion diluter
- Renewal rate of test solution (frequency/flow rate): 0.5 L every dilution cycle of 7.2 minutes
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.11 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Mains water mixed with deionised water (to give total hardness of 100 - 250 mg CaCO3/L). Mixed water passed through activated carbon filter and a UV sterilizer.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Days 0, 1, 6, 10, 15, 21 and 28

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hr light, 8 hr dark, 30 minute transition period
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality: days 0, 1, 6, 10, 15, 21 and 28; Weight: Days 0, 14, 28. Length: days 0, 28, Sub lethal effects: daily.

RANGE-FINDING STUDY: None reported

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

weight and length

Remarks on result:

other: 95% CL: 4.5 - 31 mg/L (weight), 8.5 - 27 mg/L (length)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

36 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

weight and length

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

weight and length

Details on results:

- Fish weights (individual and mean values) on day 0 and 28: see Table 2
- Tank-average or pseudo-specific growth rates for 0-28 days: see Table 3
- Type of and number with morphological abnormalities: The following sub-lethal abnormalities were observed: Unhealthy skin appearance, brown colouration, Exothalmia, increased pignmentation, loss of pigmentation, missing tail
- Type of and number with behavioural abnormalities: The following behavioural abnormalities were obsered: hyperventilation, loss of equilibrium, lethargy, uneaten food, unusual positioning, unusual swimming.
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: An air lock in the dosing tube on day 23 was rectified immediately.

Reported statistics and error estimates:

A two-tailed Dunnet's test was used to determine whether effects in the treatment groups were significantly different to the controls (5 % type one error rate). Sharpio-Wilk's test for normality and Levene's test for homogenity of variance (SAS 2002-2003) were used to determine whether a transformation of the data was required.
EC20 calcualted by fitting Chapman 1998 concentration -response model to data using the NEWTON iterative method of paramter estimation with SAS procedute NLIN. Weighting was performed as per guideline (OECD 215, 2000)

Table 2: Weight and length of test organisms

Treatment	Rep	Weight (g)		Length (cm)
		Day 0	Day 28	Day 0	Day 28
mg/L
Control	1	1.08	2.63	5	6.5
	2	1.17	2.11	5.3	6
	3	1.13	3.34	5.1	7
	4	1.33	2.94	5.3	6.8
	5	1.27	3.31	5.2	6.9
	6	1.17	4.07	4.7	7.5
	7	1.08	2.66	4.8	6.6
	8	1.18	3.02	5.1	6.9
	9	1.2	3.25	5.3	6.9
	10	1.15	3.95	5	7.7
	Mean	1.176	3.128	5.08	6.88
1.2	1	1.09	5.44	4.9	8.1
	2	1.31	3.51	5.5	7
	3	1.07	2.57	5	6.1
	4	1.07	2.49	5.1	6.3
	5	1.06	3.77	5	7.3
	6	1.06	4.94	4.9	8
	7	1.19	3.61	5.2	7
	8	1.28	3	5.3	6.8
	9	1.28	3.25	5.2	7
	10	1.3	4.02	5.2	7.4
	Mean	1.171	3.66	5.13	7.1
1.5	1	1.24	3.57	4.8	7
	2	1.16	3.82	5.2	7.2
	3	1.09	3.3	5.1	6.8
	4	1.07	4.57	4.8	7.6
	5	1.31	3.58	5.4	7
	6	1.11	2.55	5.1	6.4
	7	1.04	2.52	4.9	6.4
	8	1.09	4.1	5	7.5
	9	1	3.56	4.9	7
	10	1	2.27	4.9	6.1
	Mean	1.111	3.384	5.01	6.9
3.4*	1	1.06	3.85	5	7.3
	2	1	2.83	4.8	6.6
	3	1.05	4.75	5	7.8
	4	1.05	3.26	4.9	6.8
	5	1.15	2.92	5	6.7
	6	1.09	2.49	5	6.2
	7	1.02	2.46	4.8	6.2
	8	1.01	4.04	5	7.6
	9	1.03	dead	4.9	dead
	10	1.13	dead	5.1	dead
	Mean	1.059	3.325	4.95	6.9
8*	1	1.11	2.65	5	6.5
	2	1.15	4.85	5	7.8
	3	1.07	3.45	5.1	6.8
	4	1.26	2.39	5.3	6.4
	5	1.32	3.15	5.3	6.8
	6	1.17	3	5.1	6.5
	7	1.16	3.85	5.1	7.4
	8	1.13	2.76	5.2	6.7
	9	1.04	dead	5	dead
	10	1.06	terminated	4.9	terminated
	Mean	1.147	3.2625	5.1	6.8625
19	1	1.9	2.34	5.2	6.4
	2	1.1	2.76	5	6.6
	3	1.04	2.74	4.9	6.6
	4	1.22	1.25	5.2	5.3
	5	1	3.52	4.9	7.2
	6	1	3.78	4.5	7.3
	7	1.25	1.34	5.4	5.2
	8	1.03	3.44	5	7
	9	1.07	2.44	5	6.2
	10	1.09	terminated	5	terminated
	Mean	1.17	2.623333	5.01	6.422222
36	1	1.23	1.2	5.2	5.6
	2	1.1	1.47	4.9	5.5
	3	1.16	1.84	5.1	5.9
	4	1.38	1.87	5.2	7
	5	1.15	1.62	4.9	5.5
	6	1.21	1.83	5	5.9
	7	1.18	1.49	5.1	5.4
	8	1.23	1.6	5.2	5.5
	9	1.07	1.75	5.1	5.8
	10	1	escaped	4.9	escaped
	Mean	1.171	1.63	5.06	5.788889

Table 3: Growth rates

Mean measured concentration (mg/L)	Growth rate Day 0 - 28
	Weight	Length
Control	3.4	1.1
1.2	4	1.2
1.5	3.9	1.1
3.4	4.0*	1.2*
8	3.7*	1.1*
19	2.9	0.87
36	1.2	0.47

*Treatments 3.4 and 8.0 mg/L excluded from analyses as mortality exceeded 10%

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations the 28-day EC20 for growth rate for weight and length is 18 mg/L (95% CL: 4.5 -31 mg/L (weight), 8.5 - 27 mg/L (length)), the LOEC is 36 mg/L and the NOEC is 19 mg/L.

Executive summary:

The effect of the test substance on the growth of the juvenile rainbow trout (Oncorhynchus mykiss) was determined. In accordance with the guidelines, fish were exposed to the following range of nominal concentrations of 0.78, 1.7, 3.8, 8.3, 18, 40 mg/L. The resultant mean measured concentrations, calculated from the analysed concentrations at 0, 7, 14, 21 and 28, were 1.2, 1.5, 3.4, 8.0, 19, 36 mg/L. Results were compared to a dilution water control. Based on the mean measured concentrations the 28-day EC20 for growth rate for weight and length is 18 mg/L (95% CL: 4.5 -31 mg/L (weight), 8.5 - 27 mg/L (length)), the LOEC is 36 mg/L and the NOEC is 19 mg/L.

Description of key information

NOEC=19 mg/L, 28d, flow-through, OECD 215, Albuquerque 2005

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

19 mg/L

Additional information

The effect of the test substance on the growth of the juvenile rainbow trout (Oncorhynchus mykiss) was determined. In accordance with the guidelines, fish were exposed to the following range of nominal concentrations of 0.78, 1.7, 3.8, 8.3, 18, 40 mg/L. The resultant mean measured concentrations, calculated from the analysed concentrations at 0, 7, 14, 21 and 28 days, were 1.2, 1.5, 3.4, 8.0, 19, 36 mg/L. Results were compared to a dilution water control. Based on the mean measured concentrations the 28-day EC20 for growth rate for weight and length was 18 mg/L (95% CL: 4.5 -31 mg/L (weight), 8.5 - 27 mg/L (length)). The LOEC was 36 mg/L and the NOEC was 19 mg/L. The study is considered to be reliable and adequate for the purposes of risk assessment, classification and labelling.