2-chlorotoluene

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-11-22 to 2001-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

High Performance Liquid Chromatography (HPLC)

Details on sampling:

For each test vessel in all test groups, 0.75 mL of each test solution before and after water change was taken three times during the exposure period, equal amounts of acetonitrile were added, mixed, and then analyzed by HPLC. The test substance of each test solution was quantified from the ratio with the peak area of the standard solution.

Vehicle:

yes

Remarks:

2-methoxyethanol and HCO-60

Details on test solutions:

Preparation of the solution
The diluted water used to prepare the test solution was aerated before preparation and set to 20 ± 1 °C in a constant temperature bath. 46.2 μL (50 mg) of the test substance was collected, mixed with 100 mg of
2-methoxyethanol and 200 mg of HCO-60, diluted with purified water, and the volume was adjusted to 500 mL. At the same time, a concentrated solvent control solution (2-methoxyethanol 200 mg/L, HCO-60 400 mg/L) containing no test substance was prepared.
Dilution water was filled in a 1.0 L volumetric flask and the above concentrated test substance solution was added according to each concentration, and at the same time, the stock solution was concentrated so that the solvent concentration becomes constant (6 mg/L) in all concentration groups. 80 mL each was placed in 4 test vessels per concentration.
As solvent control diluted water containing only the test substance having the same concentration (6 mg/L) as the test concentration group, was used. Diluted water containing no test substance or solvent was used as the control group. The state (appearance) of the test solution at the time of preparation was colorless and transparent in all test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

1) Japanese name: Daphnia magna
2) Scientific name: Daphnia magna
3) Obtained from: National Environmental Research Institute,
Environment Agency (currently National Environmental Research Institute, Ministry of the Environment)
4) Obtained date: July 18, 1995
5) Sensitivity: Reference substance (Half-swimming inhibition concentration (EiC50) for 48 hours by potassium dichromate
(special grade reagent) = 0.68 mg/L (This value is the EiC50 value of 0.57 to app. 1.02 mg/L since June 1998 at institute, n=5)
6) Growth stage: Female neonate (within 24 hours)
7) Breeding conditions for parent Daphnia magna to obtain test organisms:
Breeding water: Diluted water (see 3.2)
Breeding density: 1 female/80 mL breeding water (25 females/2 L)
Water temperature: 20 ± 1 °C
Dissolved oxygen concentration: 60% or more of saturation concentration
pH: 6.7 to approx. 8.5
Lighting: Room light, 16 hours bright (800 lux or less)/8 hours dark
Breeding period: January 24, 2001-February 15, 2001
Parent mortality rate for 2 weeks
before the start of exposure: <5%
Occurrence of
dormant eggs and males: None
Type of food: Chlorella vulgaris (unicellular green algae) (Use by centrifuging the culture solution from living algae and replacing it with diluted water)
Feeding amount: 0.2 mg C (organic carbon content)/daphnid/day
Replacement of breeding water: Regularly exchanged 3 times/week

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Total hardness was 205 to 250 mg/L (CaCO3 equivalent)

Test temperature:

19.7 to 20.5 °C

pH:

7.3 to 8.2

Dissolved oxygen:

- 7.6 to 8.8 mg/L
saturated dissolved oxygen concentration at 20.0 °C: 8.8 mg/L

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

Test concentration (n): 0.0300, 0.0700, 0.170, 0.420, 1.00 mg/L (common ratio: about 2.4)

Details on test conditions:

I. Test conditions
1) Exposure method: Semi-static (replace the entire amount of test solution after 24 hours),
cover the water surface with Teflon sheet)
2) Exposure period: 21 days
3) Test water volume: 80 mL
4) Number of stations: 10 vessels/experimental design
5) Number of organisms: 10 neonates/1 concentration group (1 neonate/vessel)
6) Test water temperature: 20+1 °C
7) Dissolved oxygen concentration: 3 mg/L or more, preferably 60% or more of the saturation concentration.
Do not aerate the test solution during the exposure period
8) pH: 6-9 (This does not apply if it is caused by the test substance), fluctuation within 1.5. However, the pH of the test solution is not adjusted.
9) Hardness: Approximately 250 mg/L (CaCO3 equivalent)
10) Illumination: 16 hours bright (800 lux or less)/8 hours dark
11) Feeding: Type: Chlorella vulgaris (Use by centrifuging the culture solution from living algae and replacing
it with diluted water); Amount: 0.15 mgC (organic carbon content)/neonate/day

II. Dilution water
Use the M4-medium according to Elendt in Appendix (Appendix 1) of OECD Revised Guidelines No. 211. The composition is shown in Appendix 1.

III Observation of daphnids:
- Parental Daphnia: Life and death, swimming behaviour, and appearance abnormalities were observed and recorded daily.
Any dead individuals were removed.
- Living offspring: The number of living offspring was counted and removed from the first offspring every day.
The presence or absence of dead offspring, aborted eggs, and dormant eggs was observed and
recorded. The first brood date (first brood date) was recorded.

If daphnia could not be swam for 15 seconds after being gently moved, it was considered that swimming was inhibited (however, swimming means that it could swim in water, and those that crawl on the bottom of the water were included in the inhibition). For those, if the daphnid is forcibly submerged in water by an operation such as dropping water droplets, it may swim, but if it resurfaces, it is included in the swimming inhibition. Also, even if it is not normal swimming, it takes 15 seconds. If the neonate swam in the water even once, it was not included in the inhibition).

IV Water quality measurement:
Water temperature, dissolved oxygen concentration, pH and hardness were measured four times during the exposure period, before
and after water change, for each test vessel in all test groups.

V. Test concentration setting
Results of acute swimming inhibition test on Daphnia magna (48-hour EiC50 value based on nominal concentration. Based on 0.884 mg/L) were taken into account for the determination of test concentrations.
The test level concentrations were determined as follows:
Control area, solvent control group (no test substance added);
0.0300, 0.0700, 0.170, 0.420, 1.00 mg/L (geometric spacing: about 2.4)
The highest level is above the observed EC50 (measured concentration) in the acute test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (special grade reagent)

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.352 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

50% reproductive inhibition concentration

Remarks on result:

other:

Remarks:

(95%-confidence limit: (95%-confidence limit: cannot be calculated)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.313 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.744 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.481 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

median lethal concentration (LC50) of parental Daphnia

Remarks on result:

other:

Remarks:

(95%-confidence limit: 0.313-0744 mg/L)

Results with reference substance (positive control):

48 hours EiC50 value of 0.57 to approx. 1.02 mg/L

Reported statistics and error estimates:

For statistical analysis, Yukms software Statlight [comparison of multiple groups] (Yukms Corp, Tokyo) was used.

Statistical Method employed: Bartlett Homoscedasticity Test, One-Way Analysis of Variance (ANOVA)
(Dunnett, Williams, and Scheffes multiple comparison test was not performed because no significant difference was found as a result of one-way ANOVA.)

Table 1: Measured concentration of the test substance in test water during a 21 -day exposure period (Daphnia reproduction inhibition test under semi-static test conditions)

Nominal concentration [mg/L]	Date	Measured Concentration [mg/]					TWM* [mg/L]		% of nominal
	0 new	1 old	7 new	8 old	14 new	15 old
Control	<0.0005	<0.0005	<0.0005	<0.0005	<0.0005	<0.0005	---	---
Solvent Control	<0.0005	<0.0005	<0.0005	<0.0005	<0.0005	<0.0005	---	---
0.0300	0.0259	0.0201	0.0224	0.0186	0.0242	0.0202	0.0218	73
0.0700	0.0595	0.0496	0.0474	0.0401	0.0558	0.0503	0.0504	72
0.170	0.153	0.128	0.145	0.121	0.143	0.120	0.135	79
0.420	0.367	0.295	0.330	0.272	0.352	0.269	0.313	75
1.00	0.847	0.685	0.783	0.680	0.811	0.669	0.744	74

*: Time-weighted mean mesured concentration during 21 -days

new: freshly prepared test solution
old: old test solution before renewal

Table 2: Measured concentration as percentage of nominal

Nominal concentration [mg/L]	Date	Measured Concentration as percentage of nominal
	0 new	1 old	7 new	8 old	14 new	15 old
0.0300	86	67	75	62	81	67
0.0700	85	71	68	57	80	72
0.170	90	75	85	71	84	71
0.420	87	70	79	65	84	64
1.00	85	69	78	68	81	67

new: freshly prepared test solution
old: old test solution before renewal

	Concentration [mg/L]		% of nominal
	min.	max.	min.	max.
New	0.0224	0.847	68	90
old	0.0186	0.685	57	75

Table 3: Time (days) to first brood production

Vessel No.	Nominal concentration [mg/L], (measured concentrtion [mg/L]*)
	Control	Solvent Control	0.0300 (0.0218)	0.0700 (0.0504)	0.170 (0.135)	0.420 (0.313)	1.00 (0.744)
1	8	8	8	8	8	8	8
2	8	8	8	8	8	8	8
3	8	8	8	8	8	8	8
4	8	8	8	8	8	8	9
5	8	8	8	8	8	8	8
6	8	8	8	8	8	8	12
7	8	8	8	8	8	8	8
8	8	8	8	8	8	8	8
9	8	8	8	8	8	8	11
10	8	8	8	8	8	8	8
Min	8	8	8	8	8	8	8
Max	8	8	8	8	8	8	12

* time-weighted mean measured concentration

Table 4: Mean cumulative numbers of juveniles produced per adult alive for 21 days

Nominal concentration [mg/L]	Days
	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0.0	0.0	12.1	12.2	12.2	28.0	41.8	41.8	41.8	73.1	73.1	73.1	88.8	100.0	100.0	121.0
Solvent control	0.0	0.0	13.4	13.4	13.4	45.9	46.1	46.1	53.6	79.7	79.7	82.1	111.0	111.0	111.0	134.0
0.0300	0.0	0.0	13.6	13.7	13.7	42.5	44.1	44.1	51.6	77.9	77.9	77.9	105.0	105.8	105.8	125.4
0.0700	0.0	0.0	12.6	12.6	12.6	38.9	41.1	41.1	48.7	75.5	75.5	75.5	104.0	104.0	104.0	122.5
0.170	0.0	0.0	13.9	13.9	13.9	44.3	44.3	44.3	53.7	76.6	76.7	80.0	105.2	105.2	105.2	127.2
0.420	0.0	0.0	11.7	11.7	11.7	34.0	38.4	38.4	45.7	68.9	68.9	72.4	93.9	96.3	96.3	114.7
1.00	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

- = all parental Daphnia were dead during a 21 -day testing period

Table 5: Cumulative number of dead parental Daphnia

Nominal concentration [mg/L]	Days
	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.0300	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.0700	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.170	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.420	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1.00	0	0	0	0	0	0	0	0	0	0	0	3	5	6	6	6	6	8	9	10	10	10

Table 6: Mortality (%) of parental Daphnia

Nominal Concentration [mg/L]	Days
	1	2	4	7	14	21
Control	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0
0.0300	0	0	0	0	0	0
0.0700	0	0	0	0	0	0
0.170	0	0	0	0	0	0
0.420	0	0	0	0	0	0
1.00	0	0	0	0	60	100

Validity criteria fulfilled:

yes

Conclusions:

The reproductive inhibition to daphnia was investigated in a semi static GLP study following OECD 211. After 21 days of exposure, the median effective concentration (EC50) of parent Daphnia per day was 0.352 mg/L and the NOEC with regard to reproduction (Mean cumulative number of neonates) was 0.313 mg/L. The above concentrations are based on measured time-weighted average values.

Executive summary:

In accordance with OECD TG 211 “Daphnia Magna Reproduction Test (proposed in April 1997) (1984)” the “immobilization and reproduction test for Daphnia magna in o-chlorotoluene” was performed under semi-static conditions. After measuring the water temperature, dissolved oxygen concentration (DO), pH and total hardness of the test solution, the test Daphnia was added using a glass pipette, and the time point was defined as the start of exposure. At that time, the total amount of breeding water in the pipette was set to be within 1% of the test solution volume. After that, Daphnia was transferred to a new test solution in a cocoon and bred until 21 days later. A healthy amount of food was fed daily during the exposure period. Daily observations on mortality and the presence or absence of abnormalities were recorded. The number of living offspring was counted and removed from the first offspring every day. The presence or absence of dead offspring, aborted eggs, and dormant eggs was recorded. The first brood date was recorded as well as the date of birth of the first larva. Water temperature, dissolved oxygen concentration, pH and hardness were measured four times during the exposure period, before and after water change, for each test vessel in all test groups.

The test substance concentration in the test solution was measured three times during the exposure period before and after the water change. The ratio of the measured value to the nominal value exceeded ± 20 %. Thus, the time-weighted mean of the measured values was used for the no-effective concentration and the minimum-effective concentration.

 

Results:

Mortality of parent Daphnia

The cumulative number of deaths and mortality of parental Daphnia in each test vessel during the exposure period was recorded. The mortality rate of parental Daphnia in the control group and the solvent control group was 0% at the end of exposure and met the validity criteria of 20% or less. The mortality rate in the highest concentration group was 100% at the end of exposure. The median lethal concentration (LC50) of parental Daphnia exposed for 21 days is determined as: 0.481 mg/L (95%-confidence limit: 0.313-0744 mg/L).

First Brood Production

To investigate the impact of the test substance on the reproductive behavior of the Daphnids the first brood date was evaluated. The first brood production of the parental Daphnia in the control group and the solvent control group was 8 days after the start of exposure and was judged to be within the normal range. In the highest concentration group, it was within 12 days after the start of exposure. Based on the data the 50% reproductive inhibition concentration (EC50) of 21-day exposure 0.352 mg/L.

Mean cumulative number of neonates

To investigate the impact of the test substance on the reproduction of the Daphnids also the mean cumulative number of juveniles per parent Daphnia was evaluated. In the Control group and solvent control group the mean cumulative number of juveniles per parental Daphnia in 21 days was 121 and 134, respectively. This meets the validity criteria of 60 or more juveniles. In the highest concentration group, all parental Daphnia died by the end of exposure. From the obtained data a 21d-NOEC (reproduction) of 0.313 mg/L and a 21 days LOEC: 0.744 mg/L were calculated.

Description of key information

NOEC (Daphnia magna; 21 d; reproduction; semi-static; TWM) = 0.313 mg/L;
LOEC (Daphnia magna; 21 d; reproduction; semi-static; TWM) = 0.744 mg/L = LC100 (21 d; parental);
EC50 (Daphnia magna; 21 d; reproduction; semi-static; TWM) = 0.352 mg/L (CI not possible);
LC50 (Daphnia magna; 21 d; parental mort.; semi-static; TWM) = 0.481 mg/L (95% CI: 0.313-0.744)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.313 mg/L

Additional information

The reliable and relevant key study (MOE Japan, 2001) on Daphnia Magna Reproduction was performed compliant with GLP according to OECD 211 (1998). 10 neonates (1 per vessel) were used for each test item concentration, control, and solvent control. The following nominal test item concentrations were applied: 0.0300, 0.0700, 0.170, 0.420, 1.00 mg/L. The semi-static test design implied full media exchange every 24 hours. Analytical monitoring was performed for all concentration levels and the controls in fresh an old media at on media freshly supplied on days 0, 7, and 14. Time weighted mean measured (TWM) concentrations were between 72% and 79% of nominal. Daily observation and recording of life-and-death swimming status and the presence or absence of abnormalities in appearance were recorded. Offspring produced was removed daily and living offspring counted. The presence or absence of dead offspring, aborted eggs, and dormant eggs was observed and recorded. The first brood date (first brood date) was recorded. All guideline validity criteria were met. Parental mortality occurred at the highest test item concentration, only (0.744 mg/L; 100% mortality at day 19). Reproduction was slightly impaired at the second highest concentration only (ca. -14% at nominal 0.42 mg/L, corresponding to measured 0.31 mg/L), but not at all lower concentrations. Accordingly, the following results were obtained:
NOEC (Daphnia magna; 21 d; reproduction; semi-static; TWM) = 0.313 mg/L;
LOEC (Daphnia magna; 21 d; reproduction; semi-static; TWM) = 0.744 mg/L = LC100 (21 d; parental);
EC50 (Daphnia magna; 21 d; reproduction; semi-static; TWM) = 0.352 mg/L (CI not possible);
LC50 (Daphnia magna; 21 d; parental mort.; semi-static; TWM) = 0.481 mg/L (95% CI: 0.313-0.744)

The reliable and relevant result of the key study is confirmed by not assignable (RL 4) data on Daphnia magna reproduction (21-days) determined in a flow-through system with analytically verified test item concentrations:
The NOEC in a 21-day Daphnia magna reproduction study (performed 1986) is reported to be 0.21 mg/L (NOEC; 21d; reproduction; flow-through; measured). At the next higher concentration (measured; 0.73 mg/L) 100% mortality was observed [US HPVIS; OECD SIDS, 2001].
These results are nearly equal to the results of the key study. While not assignable, these data corroborate the result of the key study.