Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

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:

From 2017-12-19 to 2018-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Adopted: 2 October 2012

Deviations:

yes

Remarks:

Twice temperature deviation, one fresh control solution exceed 30% of LOQ, two standard solutions were injected 5 times rather than 3 for stability evaluation, which all not affect the quality or integrity of the study

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: 20170214
Purity: >99%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 mg/L and control
- Sampling method:
Samples were taken for analysis from the freshly prepared solutions and the old solution at least twice a week.
On the day of analysis, the fresh solution was of 100 mL sampled directly and the rest 100 mL was stored in refrigerator (2-8 °C) for possible re-analysis and until the end of the test.
2 replicate solutions were sampled in old solution. Taking 100 mL samples to analysis and residual sample was stored in refrigerator (2-8 °C) for possible re-analysis and until the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Solutions of 10 mg/L was prepared before used, a measured weights of 0.0999 ~ 0.1008 g of test item were added to 10L water using a stirring rate at 370 ~ 450 rpm about 24 hours. The solution was filtrated with 0.45 μm membrane.
- Controls: taking the water directly

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation: less than 24 hours old, not the first brood progeny
- Source: Derived from a healthy stock (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discolored animals, etc.).
- Feeding during test
- Food type: 0.1~ 0.2 mg C/Daphnia/Day

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions: The stock animals were maintained in culture conditions (light, temperature, and medium) similar to those to be used in the test.
- Type and amount of food: concentrated algal suspension
- Light: 16 hours photoperiod daily, 8 hours dark

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

170 mg/L ~ 200 mg/L (as CaCO3)

Test temperature:

19.1 ~ 20.2 ℃

pH:

7.96 ~ 8.26

Dissolved oxygen:

8.24 ~ 9.38 mg/L

Nominal and measured concentrations:

Norminal: 10 mg/L
Measured: 0.0131 mg/L (The overall arithmetic mean of geometrical mean of each batch determined concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: 200 ~ 250 mL conical flask
- Aeration: Before usage, the water was aerated for more than 24 hours to attain dissolved oxygen saturation.
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: treated with Aquapro Pure Water System
- Total organic carbon: 0.046 mg/L, < 2 mg/L
- Intervals of water quality measurement: The indicators of the test water should be inspected externally by a qualified company once a year, there were no indicators affecting the test quality when last checked.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hr light (1000 - 1500 lux for cool white light corresponds close to the recommended light intensity 15-20 μE•m-2•s-1): 8 hr dark

EFFECT PARAMETERS MEASURED:
The offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. The presence of aborted eggs or dead offspring were recorded daily.
Mortality among the parent animals were recorded preferably daily.
Reproductive output per surviving parent animal, i.e. number of living offspring produced during the test per surviving parent, were recorded. The length of the living parent animals (i.e. body length excluding the anal spine) at the end of the test were measured.
First brood time, Juveniles of the first brood, total Juveniles of each parent, etc. were also measured or calculated.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 mg/L
- Results used to determine the conditions for the definitive study: The no observed effect concentration was about 10 mg/L (nominal concentration).

Reference substance (positive control):

yes

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.013 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Details on results:

- Toxic Signs:
During the 21 days exposure period, no death and visible toxic signs were observed in the control group and test group.
- Calculation:
Total number of juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were analysis by a two-sided Student T-test using a 5% significance level to obtain the no observed effect concentration (NOEC). The P value of above item in test group was higher than 5%, so there was no significant difference between test group and control group.
- Verification of Test Concentration:
The concentrations of test item were determined in samples of test media by HPLC-MS/MS method. The results showed that for the test solutions of 10 mg/L, the arithmetic mean determined concentration in fresh media was 0.0213 mg/L. The arithmetic mean determined concentration in old media was 0.0084 mg/L. The concentration in old solution was 39.4% of the fresh concentrations. The overall arithmetic mean of geometrical mean of each batch determined concentration was 0.0131 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
In the facility, tests with a reference substance of the potassium dichromate (K2Cr2O7) are done four times a year, which are all within the range 0.6 to 2.1 mg/L.
The Recently EC50-24 h of the potassium dichromate to Daphnia was 1.67 mg/L, 95% confidences were 1.43-1.96 mg/L

Table 1. Total Juveniles of Each Parent

Nominal Concentration (ug/L)	Determined concentration (mg/L)	Replicate	9d	10d	11d	12d	13d	14d	15d	16d	17d	18d	19d	20d	21d	Total	Mean	SD	Mean±SD	P-value	CV
control	/	1	8	/	18	/	/	31	/	24	/	24	/	30	/	135	141	10	141±10	/	0.065
		2	12	/	22	/	/	33	/	23	/	25	/	24	/	139
		3	11	/	25	/	/	35	/	22	/	25	/	27	/	145
		4	7	/	/	24	/	/	34	/	/	32	/	29	/	126
		5	7	/	19	/	23	/	/	33	/	/	35	/	29	146
		6	16	/	21	/	/	32	/	22	/	26	/	36	/	153
		7	/	9	4	/	21	/	/	35	/	30	/	35	/	134
		8	14	/	/	20	/	/	36	/	/	29	/	36	/	135
		9	9	/	27	/	/	31	/	31	/	28	/	30	/	156
		10	12	/	8	/	/	29	/	/	31	/	35	/	27	142
10	0.0131	1	/	18	/	22	/	/	36	/	31	/	/	27	/	134	142	11	142±11	0.92	0.076
		2	9	/	20	/	/	30	/	/	29	/	34	/	30	152
		3	8	/	22	/	/	29	/	28	/	31	/	26	/	144
		4	11	/	23	/	/	32	/	31	/	28	/	25	/	150
		5	7	/	/	27	/	/	38	/	/	28	/	27	/	127
		6	13	/	25	/	17	/	/	25	/	27	/	30	/	137
		7	9	/	27	/	/	30	/	/	29	/	32	/	29	156
		8	16	/	20	/	/	32	/	/	27	/	31	/	28	154
		9	8	/	/	28	/	/	32	/	/	33	/	/	32	133
		10	7	/	/	25	/	/	35	/	/	35	/	28	/	130

Table 2 Accumulated mortality and observed toxicity signs record

Nominal Concentration (mg/L)	Determined concentration (mg/L)	Replicate	Dead time (day)	Parental mortality	Mean±S.D.	P
control	/	1	ND	0%	0±0.00	/
		2	ND	0%
		3	ND	0%
		4	ND	0%
		5	ND	0%
		6	ND	0%
		7	ND	0%
		8	ND	0%
		9	ND	0%
		10	ND	0%
10	0.0131	1	ND	0%	0±0.00	N/A
		2	ND	0%
		3	ND	0%
		4	ND	0%
		5	ND	0%
		6	ND	0%
		7	ND	0%
		8	ND	0%
		9	ND	0%
		10	ND	0%

Notes: ND means not die and no toxicity sighs until the end of the test.

N/AMeans not calculated.

Table 3. Water Quality and Physicochemical Measurements in the Definitive Test

Measured item	2017-12-20	2017-12-21	2017-12-27	2017-12-28	2018-1-5	2018-1-6	2018-1-9	2018-1-10
	Freshly	Old	Freshly	Old	Freshly	Old	Freshly	Old
pH	8.16	8.24	8.06	8.15	8.08	8.04	8.06	8.01
D.O.	9.37	9.00	9.24	9.16	8.30	8.24	8.67	8.33
T	19.3	19.9	19.1	19.3	19.2	20.2	20.1	19.1
Water Hardness (mg/L)	170	180	180.0	180	190	200	180	180
pH	8.26	8.26	8.10	8.17	7.96	8.03	8.07	8.02
D.O.	9.38	8.87	9.27	9.20	8.37	8.24	8.27	8.26
T	19.4	19.8	19.2	19.2	19.2	19.9	20	19.1
Water Hardness (mg/L)	200	190	190	190	180	180	180	170

Note: T: Temperature.

Validity criteria fulfilled:

yes

Conclusions:

The 21-day NOEC for all the parameters, such as total juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were 0.0131 mg/L.

Executive summary:

A Daphnia magna Reproduction Test of the test substance was conducted with Daphnia magna over 21 days exposure period according to OECD Guideline 211.

Based on the results of the preliminary test, the definitive test was carried out at concentration of 10 mg/L (nominal concentration). The actual concentrations of test solutions were measured by HPLC-MS/MS method. A concurrent blank control was performed in parallel. 10 daphnias were used for the test group and the control group. 10 animals individually held at test concentration and control was used. Based on the results of the preliminary test, a semi-static system renewed at 24 h with solution was used in the definitive test.

 

During the 21 days exposure period, no death and visible toxic signs were observed in the control group and test group.

The concentrations of test item were determined in samples of test media by HPLC-MS/MS method. The results showed that for the test solutions of 10 mg/L, the arithmetic mean determined concentration in fresh media was 0.0213 mg/L. The arithmetic mean determined concentration in old media was 0.0084 mg/L. The concentration in old solution was 39.4% of the fresh concentrations. The overall arithmetic mean of geometrical mean of each batch determined concentration was 0.0131 mg/L.

Total number of juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were analysis by a two-sided Student T-test using a 5% significance level to obtain the no observed effect concentration (NOEC). The P value of above item in test group was higher than 5%, so there was no significant difference between test group and control group.

 

The 21-day NOEC for all the parameters, such as total juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were 0.0131 mg/L.

Description of key information

A Daphnia magna Reproduction Test of the test substance was conducted with Daphnia magna over 21 days exposure period according to OECD Guideline 211.

The 21-day NOEC for all the parameters, such as total juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were 0.0131 mg/L (the arithmetic mean of geometrical mean of each batch determined concentration).

Additional information

A Daphnia magna Reproduction Test of the test substance was conducted with Daphnia magna over 21 days exposure period according to OECD Guideline 211.

Based on the results of the preliminary test, the definitive test was carried out at concentration of 10 mg/L (nominal concentration). The actual concentrations of test solutions were measured by HPLC-MS/MS method. A concurrent blank control was performed in parallel. 10 daphnias were used for the test group and the control group. 10 animals individually held at test concentration and control was used. Based on the results of the preliminary test, a semi-static system renewed at 24 h with solution was used in the definitive test.

During the 21 days exposure period, no death and visible toxic signs were observed in the control group and test group.

The concentrations of test item were determined in samples of test media by HPLC-MS/MS method. The results showed that for the test solutions of 10 mg/L, the arithmetic mean determined concentration in fresh media was 0.0213 mg/L. The arithmetic mean determined concentration in old media was 0.0084 mg/L. The concentration in old solution was 39.4% of the fresh concentrations. The overall arithmetic mean of geometrical mean of each batch determined concentration was 0.0131 mg/L.

Total number of juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were analysis by a two-sided Student T-test using a 5% significance level to obtain the no observed effect concentration (NOEC). The P value of above item in test group was higher than 5%, so there was no significant difference between test group and control group.

The 21-day NOEC for all the parameters, such as total juveniles of each parent, body length, first brood time, batch number of each parent, Juveniles of the first brood of each parent were 0.0131 mg/L.