4,4'-cyclohexylidenedi-o-cresol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

40 mL samples were collected from each replicate test vessel after at the start and end of the test

Vehicle:

yes

Details on test solutions:

Water used for the acclimation of test organisms and for all toxicity testing was carbon filtered, deionised water collected at the testing facility. Water was adjusted to a hardness of 160 to 180 mg/L and stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet steriliser. Prior to use, the pH of the dilution water was adjusted to less than 8.0 with 5 % H₃PO₄. A sample of dilution water collected at the start of the definitive test had a hardness of 176 mg/L as CaCO₃, a particulate matter concentration less than 10 mg/L, and a total organic carbon concentration of 1.2 mg/L. The concentration of residual chlorine in the dilution water at the start of the definitive toxicity test and from one control vessel at 24 and 48 hours was always less than 0.01 mg/L.

A stock solution of DMBPC was made with a nominal concentration of 12 000 mg/L by bringing 0.6058 g of DMBPC to a total volume of 50 mL with dimethylformamide.

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile daphnids, Daphnia magna, less than 24 hours old, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were produced by an in-house culture of adult daphnids that were maintained under test conditions at the testing facility for 27 days. At the conclusion of the test the control daphnids had an average wet weight of 0.23 mg (loading rate during the definitive toxicity test was approximately 9.2 mg per liter).

Prior to testing, the daphnid culture was maintained in 100 % dilution water under static renewal conditions, and the test organisms were not treated for disease. No mortality was observed during the 48 hours prior to the beginning of the test and at the beginning of the test organisms were apparently free of disease, injuries, and abnormalities. The culture used to produce neonates for the definitive toxicity test produced young by day 8 and a subsample of adults from the culture produced an average of at least 3 young per daphnia during the 7 day period prior to the start of the test. During the 7 days prior to the beginning of the test, the temperature of the culture ranged from 19.1 to 20.1 °C, the pH ranged from 7.4 to 7.5, and the dissolved oxygen concentration was always at least 8.7 mg/L. Test organisms were fed the freshwater algae, Selenastrum capricornutum, and a mixture of yeast and trout chow once each day before the test. Daphnids were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

176 mg/L as CaCO₃ (at study start)

Test temperature:

20.0 to 21.0 °C

pH:

7.2 to 7.3

Dissolved oxygen:

8.0 to 8.6 mg/L

Nominal and measured concentrations:

Nominal: 0 (control and solvent control), 160, 260, 430, 720 and 1200 µg a.i./L
Measured: ND (not detected at or above the limit of quantitation of 8 µg a.i./L; control and solvent control), 139, 233, 384, 664 and 1084 µg a.i./L

Details on test conditions:

A range-finding test was conducted under static conditions with a control, and five concentrations of test substance: 10, 50, 100, 500, and 1200 µg/L. A second range-finding test was conducted under static conditions with the same concentrations but included a solvent control to determine if using a solvent increased solubility or toxicity. The results of both range-finding assays were identical: after 48 hours there was 100 % survival in the controls and at all tested concentrations and no sub-lethal effects were observed at any tested concentration. In the second range-finding assay, samples were collected from the control, solvent control, 10 and 1200 µg/L test vessels at the start and end of the test and were analysed by the sponsor. The resulting mean measured concentrations were ND (not detected at or above the limit of detections of 8 µg/L in the control and solvent control), 13.9 and 1020 µg/L, respectively.

The definitive toxicity test was conducted under static conditions at 20 ± 2 °C with five concentrations of test substance a dilution water control, and a solvent control (0.10 mg/L dimethylformamide). Nominal concentrations of the active ingredient were 0 (control and solvent control), 160, 260, 430, 720 and 1200 µg/L. The solvent control received 0.1 mL/L dimethylformamide, the same concentration of solvent that was delivered to test vessels containing the highest concentration of DMBPC.

Twenty daphnids were randomly and equally distributed among two replicates of each treatment and control. The test was performed in 300 mL glass beakers that contained 250 mL litres of test solution (water depth was approximately 10 cm). Test vessels were randomly arranged in a water bath during the 48 hour test (random numbers tables were used to select the location of each vessel). A 16-hour light and 8-hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 49 footcandles. A 15 minute transition period was provided between dark and light. Aeration was not required to maintain dissolved oxygen concentrations above acceptable levels. The numbers of surviving organisms, and the occurrence of sub-lethal effects and insoluble material were determined visually and recorded after 0, 24 and 48 hours. Dead test organisms were removed when first observed. Dissolved oxygen, pH, conductivity, and temperature were measured and recorded daily in each test vessel. The temperature in a representative vessel was recorded continuously during the test.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 084 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 084 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

No insoluble material was observed throughout the test.
Mean measured concentrations ranged from 87 to 92 % of nominal concentrations and they were stable throughout the test. Exposure of daphnids to DMBPC resulted in 24 and 48 hour EC50 values greater than 1084 µg/L, the highest tested concentration and the approximate test substance water solubility limit. The 48 hour NOEC is 1084 µg/L. This concentration of DMBPC allowed 95 % survival and did not cause any immobilisation or other sub-lethal effects.

During the definitive toxicity test the conductivity ranged from 590 to 650 µmhos/cm (mean = 620 µmhos/cm), the pH ranged from 7.2 to 7.3, the temperature ranged from 20.0 to 21.0 °C (mean = 20.5 °C), and the dissolved oxygen concentration ranged from 8.0 to 8.6 mg/L (mean = 8.4 mg/L).

Reported statistics and error estimates:

Results of the toxicity test could not be interpreted by standard statistical techniques, because there was 95 % survival at the highest tested concentration. The no observed effect concentration is the highest tested concentration at which there was no toxicant-related mortality, immobilisation, abnormal behavior or physiological effects.

Summary of results

Duration (hours)	Endpoint	Effect concentration (µg/L)	Nominal / Measured	Conc. Based on	Basis for Effect
48	LC50	>1084	measured	a.i.	Mortality/ Mobility
48	EC50	>1084	measured	a.i.	Mobility
48	NOEC	1084	measured	a.i.	Mobility
24	LC50	>1084	measured	a.i.	Mortality/ Mobility
24	EC50	>1084	measured	a.i.	Mobility

 

 

 

Measured concentrations of DMBPC in test media

Nominal Concentration of DMBPC (µg a.i./L)	Measured Concentration of DMBPC (µg a.i./L)
	Rep.	0 Hour	48 Hours	Mean	% Recovery
Control	1	ND	ND	ND	--
	2	ND	ND
Solvent Control	1	ND	ND	ND	--
	2	ND	ND
160	1	142	132	139	87
	2	--¹	144
260	1	236	219	233	90
	2	247	231
430	1	405	364	384	89
	2	387	379
720	1	670	654	664	92
	2	685	646
1200	1	1111	1061	1084	90
	2	1111	1054
Stock Solution
1200	--	1138	1157	1148	96
Matrix Spike Sample
430	--	420	434	412	96
	-	392	401
Laboratory Control Samples
430		428	377	403	94
Blank
0		ND	ND	ND	--
ND: none detected at or above the limit of quantitation (8.0 µg/L) ¹Sample was lost due to broken vial.

 

 

 

Survival and sublethal effect data

Mean Measured Concentration of DMBPC (µg/L)	Rep	Number Alive			Number Affected
		0 hr	24 hr	48 hr	0 hr	24 hr	48 hr
Control	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
Solvent Control	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
139	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
233	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
384	1	10	10	9	0	0	0
	2	10	10	10	0	0	0
664	1	10	10	10	0	0	0
	2	10	10	10	0	0	0
1084	1	10	10	10	0	0	0
	2	10	10	9	0	0	0
Rep = Replicate

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48 hour EC50 value was >1084 µg a.i./L. The 48 hour NOEC was 1084 µg a.i./L.

Executive summary:

The toxicity potential of the test material to the freshwater invertebrate daphnia magna was investigated in accordance with the standardised guideline EPA OPPTS 850.1010 under GLP conditions.

The definitive toxicity test was conducted under static conditions at 20 ± 2 °C with five concentrations of test substance a dilution water control, and a solvent control (0.10 mg/L dimethylformamide). Nominal concentrations of the active ingredient were 0 (control and solvent control), 160, 260, 430, 720 and 1200 µg/L. The solvent control received 0.1 mL/L dimethylformamide, the same concentration of solvent that was delivered to test vessels containing the highest concentration of DMBPC.

Twenty daphnids were randomly and equally distributed among two replicates of each treatment and control. The test was performed in 300 mL glass beakers that contained 250 mL litres of test solution. The numbers of surviving organisms, and the occurrence of sub-lethal effects and insoluble material were determined visually and recorded after 0, 24 and 48 hours.

At the 48 hour observation point, a single daphnid was found to be deceased in both the 384 and 1084 µg/L dose levels. No sub-lethal effects were noted.

Under the conditions of this study, the 48 hour EC50 value was >1084 µg a.i./L, the highest tested concentration and the approximate test substance water solubility limit. The 48 hour NOEC was 1084 µg a.i./L. This concentration of DMBPC allowed 95 % survival and did not cause any immobilisation or other sub-lethal effects.

Description of key information

Under the conditions of this study, the 48 hour EC50 value was >1084 µg a.i./L. The 48 hour NOEC was 1084 µg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 1 084 µg/L

Additional information

The toxicity potential of the test material to the freshwater invertebrate daphnia magna was investigated in accordance with the standardised guideline EPA OPPTS 850.1010 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The definitive toxicity test was conducted under static conditions at 20 ± 2 °C with five concentrations of test substance a dilution water control, and a solvent control (0.10 mg/L dimethylformamide). Nominal concentrations of the active ingredient were 0 (control and solvent control), 160, 260, 430, 720 and 1200 µg/L. The solvent control received 0.1 mL/L dimethylformamide, the same concentration of solvent that was delivered to test vessels containing the highest concentration of DMBPC.

Twenty daphnids were randomly and equally distributed among two replicates of each treatment and control. The test was performed in 300 mL glass beakers that contained 250 mL litres of test solution. The numbers of surviving organisms, and the occurrence of sub-lethal effects and insoluble material were determined visually and recorded after 0, 24 and 48 hours.

At the 48 hour observation point, a single daphnid was found to be deceased in both the 384 and 1084 µg/L dose levels. No sub-lethal effects were noted.

Under the conditions of this study, the 48 hour EC50 value was >1084 µg a.i./L, the highest tested concentration and the approximate test substance water solubility limit. The 48 hour NOEC was 1084 µg a.i./L. This concentration of DMBPC allowed 95 % survival and did not cause any immobilisation or other sub-lethal effects.