4,4'-cyclohexylidenedi-o-cresol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

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 40 to 48 mg/L and stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet steriliser. A sample of dilution water collected at the start of the definitive test had a hardness of 40 mg/L as CaCO₃, an acidity of 8 mg/L as CaCO₃, an alkalinity of 27 mg/L CaCO₃, a particulate matter concentration less than 10 mg/L, and a total organic carbon concentration of 0.8 mg/L

A stock solution of DMBPC with a nominal concentration of 12 000 mg/L was prepared by bringing 6.0362 g of the test substance to a total volume of 500 mL with dimethylformamide (solvent, DMF).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Juvenile Rainbow Trout, Oncorhynchus mykiss, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were procured from a commercial supplier. Prior to testing, the fish were maintained in a 280 liter polyethylene tank. At the conclusion of the test, the control fish had an average total length of 29 mm and an average wet weight of 0.19 g (loading rate was approximately 0.13 g/L at any time and 0.02 g/L/24 hours). During acclimation, fish were not treated for disease and they were free of apparent disease, injuries and abnormalities at the beginning of the test. During the 14-day period before the start of the test, the acclimation temperature range was 11.2 to 12.9 °C and the dissolved oxygen concentration was always at least 6.8 mg/L. Fish were fed dry commercial fish food and trout chow once or twice per day except during the 48 hours preceding the test and they were not fed during the toxicity test. Mortality during the last 48 hours of acclimation was 0 %.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

40 mg/L as CaCO₃ (at study start)

Test temperature:

11.0 to 12.6 °C

pH:

7.0 - 7.5

Dissolved oxygen:

10.4 mg/L

Nominal and measured concentrations:

Nominal: 0 (control and solvent), 160, 260, 430, 720 and 1200 µg a.i./L
Mean measured: ND (not detected at or above the limit of detection of 8 µg a.i./L; control and solvent control), 143, 258, 410, 681 and 1094 µg a.i./L

Details on test conditions:

A range-finding test with DMBPC was conducted under static conditions with a control, a solvent control, and five concentrations of test substance: 0.99, 9.9, 100, 500, and 1200 µg a.i./L. After 96 hours there was 0 % survival at 1200 µg a.i./L and 100 % survival in the control, solvent control, and at all tested concentrations. No sub-lethal effects were observed at the end of the test.

The definitive toxicity test was conducted under flow-through conditions at 12 ± 2 °C with five concentrations of test substance, a dilution water control and a solvent control (0.10 mg/L dimethylformamide). 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 rainbow trout were randomly and equally distributed among two replicates of each treatment and control. The test was performed in 20-litre glass aquaria that contained 15 litres of test solution (water depth was approximately 18 cm). Test vessels were randomly arranged in a water bath during the 96-hour test (a random numbers table was used to select the location of each vessel and add test organisms). A 16-hour light and 8-hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 38 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, the occurrence of sub-lethal effects and the presence of insoluble material were determined visually and recorded after 0, 3, 24, 48, 72 and 96 hours. Dead test organisms were removed twice each day.

The test substance was supplied to the test vessels under flow-through conditions by an intermittent flow proportional diluter. The diluter, which was constructed at the testing facility, allowed test substance to contact only glass or Teflon® surfaces. The diluter was operated from approximately 52 hours prior to the start of the definitive toxicity test, was calibrated before and after the test, and was monitored for normal operation twice per day. During the test the diluter was activated 759 times, resulting in an average of 6.3 volume additions per 24 hours in each test vessel. Flow to each replicate test vessel was within 10 % of the flow to the alternate replicate. Dissolved oxygen, pH, conductivity and temperature were measured and recorded daily in each test vessel. The temperature in a control vessel was recorded continuously during the test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

528 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL = 410 to 681 µg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

143 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and sublethal effects

Details on results:

No insoluble material was observed in any test vessel during the test.
The mean measured concentrations were 89 to 99 % of the nominal concentrations and were stable during the test. One hundred percent survival occurred in the control and solvent control, and no sub-lethal effects were noted in either of these controls throughout the test.

During the definitive toxicity test the conductivity ranged from 140 to 150 µmhos/cm (mean = 140 µmhos/cm), the pH ranged from 7.0 to 7.5, the temperature ranged from 11.0 to 12.6 °C (mean = 11.6 °C), and the dissolved oxygen concentration ranged from 10.2 to 10.8 mg/L (mean = 10.4 mg/L).

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

Results of the toxicity test were interpreted by standard statistical techniques when warranted. The 24 hour LC50 value could not be calculated because there was greater than 50 % survival of trout at all test concentrations. The binomial method was used to calculate the 48, 72 and 96 hour LC50 values. The slope of the 96 hour concentration-response curve could not be calculated with this data set. The no observed effect concentration is the highest tested concentration at which there was no observed mortality, abnormal behavior or physiological effect.

Sublethal observations / clinical signs:

Results

Duration (hours)	Endpoint (EC50, LC50, other, etc.)	Effect conc. (µg/L)	95% Confidnece Interval	Nominal / Measured	Conc. Based on	Basis for Effect
24	LC50	>1094	--	measured	a.i.	mortality
48	LC50	837	681 to 1094	measured	a.i.	mortality
72	LC50	681	410 to 1094	measured	a.i.	mortality
96	LC50	528	410 to 681	measured	a.i.	mortality
96	NOEC	143	--	measured	a.i.	mortality

 

 

 

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	96 Hours	Mean	% Recovery
Control	1	ND	ND	ND	--
	2	ND	ND
Solvent Control	1	ND	ND	ND	--
	2	ND	ND
160	1	141	146	143	89
	2	140	143
260	1	254	262	258	99
	2	253	262
430	1	404	413	410	95
	2	405	418
720	1	676	689	681	95
	2	671	686
1200	1	1092	1103	1094	91
	2	1091	1089
Stock Solution
1200		1044	1068	1056	88
Matrix Spike Samples
430		421	422	422	98
		418	426
Laboratory Control Sample
430		427	427	427	99
Blank
0		ND	ND	ND	--
Rep = Replicate ND: none detected at or above the limit of detection (8.0 µg/L)

 

 

 

Survival and sublethal effect data

Mean Measured Conc. of DMBPC (µg a.i./L)	Rep	Number Alive					Number Affected¹
		0² hr	24 hr	48 hr	72 hr	96 hr	0² hr	24 hr	48 hr	72 hr	96 hr
Control	1	10	10	10	10	10	0	0	0	0	0
	2	10	10	10	10	10	0	0	0	0	0
Solvent Control	1	10	10	10	10	10	0	0	0	0	0
	2	10	10	10	10	10	0	0	0	0	0
143	1	10	10	10	10	10	0	0	0	0	0
	2	10	10	10	10	10	0	0	0	0	0
258	1	10	10	10	10	10	0	0	0	0	0
	2	10	10	10	10	10	0	0	0	4	3
410	1	10	10	10	10	10	0	0	2	8	9
	2	10	10	10	10	10	0	0	3	5	9
681	1	10	10	9	6	0	0	3	9	6	--
	2	10	10	10	4	0	0	4	10	4	--
1094	1	10	10	0	0	0	0	10	--	--	--
	2	10	10	0	0	0	0	10	--	--	--
Rep = Replicate ¹Affected fish were lethargic, immobilised, exhibiting a loss of equilibrium and/or exhibiting a change in colour. ²No mortality or sublethal effects were observed at 3 hours.

 

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 was 528 µg a.i./L with a 95 % confidence interval of 410 to 681 µg a.i./L. The 96 hour NOEC was 143 µg a.i./L.

Executive summary:

The toxicity potential of the test material to the freshwater fish Oncorhynchus mykiss was investigated in accordance with the standardised guideline EPA OPPTS 850.1075 under GLP conditions.

The definitive toxicity test was conducted under flow-through conditions at 12 ± 2 °C with five concentrations of test substance, a dilution water control and a solvent control (0.10 mg/L dimethylformamide).  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. Nominal concentrations were 0 (control and solvent), 160, 260, 430, 720 and 1200 µg a.i./L.

Twenty rainbow trout were randomly and equally distributed among two replicates of each treatment and control. The test was performed in 20-liter glass aquaria that contained 15 litres of test solution.

The numbers of surviving organisms, the occurrence of sub-lethal effects and the presence of insoluble material were determined visually and recorded after 0, 3, 24, 48, 72 and 96 hours. Dead test organisms were removed twice each day. 

Mean measured concentrations were: not detected at or above the limit of detection in the control and solvent control, 143, 258, 410, 681 and 1094 µg a.i./L

No mortality was observed for the first 48 hours. After 48 hours all fish in the top dose level were deceased. One fish in the 681 µg a.i./L dose group was also deceased. After 72 hours, the total number of dead fish at the 681 µg a.i./L dose level was 10. At 96 hours, all fish at this dose level were found to be deceased.

The 96 hour LC50 was 528 µg a.i./L with a 95 % confidence interval of 410 to 681 µg a.i./L. The 96 hour NOEC was 143 µg a.i./L; this concentration of DMBPC resulted in 100 % survival and did not cause any sub-lethal effects. 

Description of key information

The 96 hour LC50 in rainbow trout was 528 µg a.i./L with a 95 %. The 96 hour NOEC was 143 µg a.i./L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

528 µg/L

Additional information

The toxicity potential of the test material to the freshwater fish Oncorhynchus mykiss was investigated in accordance with the standardised guideline EPA OPPTS 850.1075 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 flow-through conditions at 12 ± 2 °C with five concentrations of test substance, a dilution water control and a solvent control (0.10 mg/L dimethylformamide).  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. Nominal concentrations were 0 (control and solvent), 160, 260, 430, 720 and 1200 µg a.i./L.

Twenty rainbow trout were randomly and equally distributed among two replicates of each treatment and control. The test was performed in 20-liter glass aquaria that contained 15 litres of test solution.

The numbers of surviving organisms, the occurrence of sub-lethal effects and the presence of insoluble material were determined visually and recorded after 0, 3, 24, 48, 72 and 96 hours. Dead test organisms were removed twice each day. 

Mean measured concentrations were: not detected at or above the limit of detection in the control and solvent control, 143, 258, 410, 681 and 1094 µg a.i./L

No mortality was observed for the first 48 hours. After 48 hours all fish in the top dose level were deceased. One fish in the 681 µg a.i./L dose group was also deceased. After 72 hours, the total number of dead fish at the 681 µg a.i./L dose level was 10. At 96 hours, all fish at this dose level were found to be deceased.

The 96 hour LC50 was 528 µg a.i./L with a 95 % confidence interval of 410 to 681 µg a.i./L. The 96 hour NOEC was 143 µg a.i./L; this concentration of DMBPC resulted in 100 % survival and did not cause any sub-lethal effects.