4,4'-cyclohexylidenedi-o-cresol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Collected from each replicate test vessel at the start and end of the test.

Vehicle:

yes

Details on test solutions:

Water used for acclimation of test organisms and for all toxicity testing was sterile freshwater AAP medium (U.S. EPA, 1978) at a pH of 7.5 ± 0.1 (the pH was adjusted with NaOH). The vehicle used was dimethylformamide (DMF).

A series of solutions were prepared by bringing 0.0377, 0.0754, 0.1509, 0.3018 and 0.6038 g of test substance to 50 mL of dimethylformamide in volumetric flasks. The solutions were shaken vigorously until the test substance dissolved. Test solutions were prepared by combining 0.050 mL of each stock solution and dilution water in 500 mL volumetric flasks. A stability blank sample was set up at 1200 µg/L and placed among the test vessels. No algae were inoculated into the stability blank.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Algae used for the test: Selenastrum capricornutum, UTEX 1648.
The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. During the 14-day acclimation period, the culture was actively growing in at least 2 subcultures prior to the start of the toxicity test. The subsample of algae used to inoculate media at the start of the definitive test came from a seven day old culture. Identification of the culture organisms, which are also referred to as Raphidocelis subcapitata, was verified using an appropriate taxonomic key.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Test temperature:

24.3 to 24.7 °C

pH:

7.2 to 7.3 (initial); 7.8 to 9.4 (termination)

Nominal and measured concentrations:

Nominal: 0 (control and solvent control), 75, 150, 300, 600 and 1200 µg/L
Mean Measured: ND (none detected; control and solvent control), 81, 161, 297, 595 and 1165 µg/L

Details on test conditions:

A range-finding test was conducted under static conditions with a control, a solvent control, and five concentrations of DMBPC: 0.0, 0.99, 9.9, 100, 500 and 1200 µg/L. At the conclusion of the test, the number of cells/mL in the test vessels equalled the following percent of the number of cells/mL in the control vessels: 0.99 g/L = 81 %; 9.9 µg /L = 79 %; 100 µg/L = 83 %; 500 µg/L = 64 %; and 1200 µg/L = 45 %.

The definitive toxicity test was conducted at 24 ± 2°C with five concentrations of test substance, a control, and a solvent control (0.10 mL/L dimethylformamide). A series of solutions were prepared by bringing 0.0377, 0.0754, 0.1509, 0.3018 and 0.6038 g of test substance to 50 mL of dimethylformamide in volumetric flasks. The solutions were shaken vigorously until the test substance dissolved.

These stock solutions were used to prepare the nominal test solutions by transferring the appropriate amount of stock solution into DMF. Water quality measurements were made, analytical samples were collected and each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. The flasks were inoculated with approximately 10 000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator during the test (a random numbers table was used to select the location for each vessel). A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 380 to 400 footcandles.

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer at 24, 48, 72, and 96 hours.

Temperature of the incubator was measured and recorded daily and the temperature in a representative vessel of water incubated with the test vessels was continuously recorded. The pH of test solutions was measured and recorded in each test solution prior to distribution to the test vessels at the start of the test and in each test vessel at the end of the test.

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (1200 µg/L nominal DMBPC concentration) was combined in a 250 mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 48 hours.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 1 165 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95 % CL = 1029 to >1165 µg/L

Duration:

96 h

Dose descriptor:

EC90

Effect conc.:

> 1 165 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 95 % CL = 1094 to >1165 µg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 1 165 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL > 1165 µg/L

Duration:

96 h

Dose descriptor:

EC90

Effect conc.:

> 1 165 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL .1165 µg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1 141 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: area under the curve

Remarks on result:

other: 95 % CL = 837 to >1165 µg/L

Duration:

96 h

Dose descriptor:

EC90

Effect conc.:

> 1 165 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: area under the curve

Remarks on result:

other: 95 % CL > 1165 µg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

595 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

161 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: area under the curve

Details on results:

Insoluble material was not observed during the test.
The initial measured concentrations ranged from 97 to 108 % of the nominal concentrations and the final measured concentrations were 57 to 76 % of the nominal concentrations.

The algal population grew well, resulting in an average of 4 173 000 cells/mL in the control after 96 hours. No effects (relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test. Water quality throughout the test was within acceptable limits. The incubator temperature ranged from 24.3 to 24.7 °C. The pH was not affected by the test substance.

Exposure of Selenastrum capricornutum to DMBPC for 96 hours resulted in an EC50 greater than 1165 µg/L when calculated using the number of cells/mL or the average specific growth rate, and 1141 µg/L when calculated using the area under the growth curve. The 96 hour NOEC is 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate, at 161 µg/L when determined using the area under the growth curve.

At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 1200 g/L) was combined with 100 mL of fresh media in a 250 mL flask. This culture was incubated under test conditions for 48 hours. During this period the number of algal cells increased from an initial calculated concentration of approximately 36 000 cells/mL to 676 000 cells/mL, indicated that any toxic effect at this concentration was algistatic rather than algicidal.

Reported statistics and error estimates:

The average specific growth rate was calculated as the natural log of the number of cells/mL at time t1 minus the natural log of the number of cells/mL at time t0 divided by the exposure period. The area under the growth curve was calculated as:

Area = (N1 - N0) / 2) x [(t1 + (N1 + N2 - 2N0) / 2)] x [(t2 - t1) + (Nn - 1 + Nn - 2N0) / 2] x (tn - tn - 1);

where: N0 = nominal number of cells/mL at time t0, N1 = measured number of cells/mL at time t1, Nn = measured number of cells/mL at time tn, t1 = time of first measurement after the beginning of the test and tn = time of nth measurement after the beginning of the test.

The EC10, EC50 and EC90 values were calculated using a weighted least squares non linear regression technique when warranted.

The no observed effect concentration (NOEC) was determined using a one-way analysis of variance (ANOVA) and Bonferroni’s test (TOXSTAT 3.3). The effective concentrations and NOECs were determined using the initial measured concentrations of DMBPC and the number of cells/mL, average specific growth rate, and area under the growth curve. Treatment data were compared to pooled control and solvent control data for the determination of the NOECs (mean control and solvent control data were never significantly different).

Effective concentrations (EC10, EC50 and EC90 values) from the toxicity test with DMBPC and the freshwater alga, Selenastrum capricornutum

Duration	Endpoint	Effect conc. (microg/L)	Nominal/ Measured	Conc. Based on (nominal, a.i., test material)	Basis for Effect	95% CL (microg/L)
48 hours	EC10	178	measured	a.i.	Cell number	<81 to 505
48 hours	EC50	>1165	measured	a.i.	Cell number	800 to >1165
48 hours	EC90	>1165	measured	a.i.	Cell number	>1165
72 hours	EC10	--	measured	a.i.	Cell number	--
72 hours	EC50	530	measured	a.i.	Cell number	248 to 1130
72 hours	EC90	>1165	measured	a.i.	Cell number	947 to >1165
96 hours	EC10	--	measured	a.i.	Cell number	--
96 hours	EC50	>1165	measured	a.i.	Cell number	1029 to >1165
96 hours	EC90	>1165	measured	a.i.	Cell number	1094 to >1165
48 hours	EC10	>1165	measured	a.i.	Growth rate	--
48 hours	EC50	>1165	measured	a.i.	Growth rate	>1165
48 hours	EC90	>1165	measured	a.i.	Growth rate	>1165
72 hours	EC10	>1165	measured	a.i.	Growth rate	--
72 hours	EC50	>1165	measured	a.i.	Growth rate	>1165
72 hours	EC90	>1165	measured	a.i.	Growth rate	>1165
96 hours	EC10	>1165	measured	a.i.	Growth rate	--
96 hours	EC50	>1165	measured	a.i.	Growth rate	>1165
96 hours	EC90	>1165	measured	a.i.	Growth rate	>1165
48 hours	EC10	--	measured	a.i.	AUC	--
48 hours	EC50	>1165	measured	a.i.	AUC	930 to >1165
48 hours	EC90	>1165	measured	a.i.	AUC	>1165
72 hours	EC10	--	measured	a.i.	AUC	--
72 hours	EC50	653	measured	a.i.	AUC	360 to >1165
72 hours	EC90	>1165	measured	a.i.	AUC	>1165
96 hours	EC10	--	measured	a.i.	AUC	--
96 hours	EC50	1141	measured	a.i.	AUC	837 to >1165
96 hours	EC90	>1165	measured	a.i.	AUC	>1165
AUC = Area under the curve

 

 

 

 

Measured concentrations of DMBPC in test media during the toxicity test with the freshwater alga,  Selenastrum capricornutum.

Nominal Concentration of DMBPC (microg/L)	Measured Concentrations of DMBPC (microg/L)
	0 Hour	% Recovery	96 Hours	% Recovery
Test Media Samples
Control	ND	--	ND	--
Solvent Control	ND	--	ND	--
75	81	108	43	57
150	161	107	98	65
300	297	99	228	76
600	595	99	435	73
1200	1165	97	900	75
Laboratory Control Sample
300	311	104	317	106
Matrix Spike Blank
300	--	--	285	95
	--	--	277	92
Stability Blank
1200	1181	98	1036	86
Blank
0	ND	--	ND	--
ND = none detected at or above 8.0 microg/L, the limit of detection.

 

 

 

 

Cell growth data from the toxicity test with DMBPC and the freshwater alga,  Selenastrum capricornutum.

Initial Measured Concentration of DMBPC (microg/L)	Rep	Number of Cells per Milliliter
		Hour of Exposure
		0	24	48	72	96
Control	1	10,000	37,000	174,000	1,290,000	4,920,000
	2	10,000	36,000	150,000	2,020,000	3,740,000
	3	10,000	29,000	170,000	1,220,000	3,860,000
	Mean	10,000	34,000	165,000	1,510,000	4,173,000
Solvent Control	1	10,000	34,000	136,000	880,000	4,420,000
	2	10,000	45,000	130,000	700,000	4,820,000
	3	10,000	35,000	156,000	960,000	4,880,000
	mean	10,000	38,000	141,000	847,000	4,707,000
	% control	100	112	85	56	113
81	1	10,000	43,000	146,000	840,000	5,360,000
	2	10,000	31,000	188,000	860,000	3,220,000
	3	10,000	23,000	140,000	720,000	4,100,000
	mean	10,000	32,000	158,000	807,000	4,227,000
	% control	100	94	96	53	101
161	1	10,000	24,000	146,000	772,000	4,640,000
	2	10,000	47,000	142,000	1,260,000	4,720,000
	3	10,000	36,000	150,000	720,000	4,400,000
	mean	10,000	36,000	146,000	917,000	4,587,000
	% control	100	106	88	61	110
297	1	10,000	26,000	124,000	720,000	3,440,000
	2	10,000	49,000	126,000	780,000	3,760,000
	3	10,000	47,000	150,000	940,000	3,720,000
	mean	10,000	41,000	133,000	813,000	3,640,000
	% control	100	121	81	54	87
595	1	10,000	36,000	100,000	448,000	3,820,000
	2	10,000	43,000	116,000	830,000	4,660,000
	3	10,000	40,000	66,000	430,000	3,640,000
	mean	10,000	40,000	94,000	569,000	4,040,000
	% control	100	118	57	38	97
1165	1	10,000	24,000	82,000	304,000	2,280,000
	2	10,000	23,000	98,000	356,000	2,860,000
	3	10,000	25,000	76,000	294,000	2,140,000
	mean	10,000	24,000	85,000	318,000	2,427,000
	% control	100	71	52	21	58
Rep = Replicate

 

 

 

 

Average specific growth rate from the toxicity test with DMBPC and the freshwater alga,  Selenastrum capricornutum.

Measured Concentration of DMBPC (microg/L)		Average Specific Growth Rate
		Hour of Exposure
		24	48	72	96
Control	mean	0.051	0.058	0.069	0.063
Solvent Control	mean	0.055	0.055	0.062	0.064
	% control	108	95	90	102
81	mean	0.048	0.057	0.061	0.063
	% control	94	98	88	100
161	mean	0.051	0.056	0.062	0.064
	% control	100	97	90	102
297	mean	0.057	0.054	0.061	0.061
	% control	112	93	88	97
595	mean	0.057	0.046	0.056	0.062
	% control	112	79	81	98
1165	mean	0.037	0.045	0.048	0.057
	% control	73	78	70	90

Limit of detection was 8.0 microg/L

 

 

 

Area under the growth curve from the toxicity test with DMBPC and the freshwater alga,  Selenastrum capricornutum.

 

Measured Concentration of DMBPC (microg/L)		Area Under the Growth Curve
		Hour of Exposure
		24	48	72	96
Control	mean	288,000	2,436,000	22,296,000	90,252,000
Solvent Control	mean	336,000	2,244,000	14,100,000	80,268,000
	% control	117	92	63	89
81	mean	264,000	2,304,000	13,644,000	73,812,000
	% control	92	95	61	82
161	mean	312,000	2,256,000	14,772,000	80,580,000
	% control	108	93	60	89
297	mean	372,000	2,220,000	13,332,000	66,528,000
	% control	129	91	60	74
595	mean	360,000	1,728,000	9,440,000	64,512,000
	% control	125	71	42	71
1165	mean	168,000	1,236,000	5,832,000	38,532,000
	% control	58	51	26	43

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 96 hour ErC50 was > 1165 µg/L. The 96 hour NOEC was 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate.

Executive summary:

The toxicity potential of the test material to freshwater algae was investigated in accordance with the standardised guideline EPA OPPTS 850.5400 under GLP conditions.

The definitive toxicity test was conducted at 24 ± 2°C with five concentrations of test substance, a control, and a solvent control (0.10 mL/L dimethylformamide). Nominal concentrations were 0 (control and solvent control), 75, 150, 300, 600 and 1200 µg/L. Each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. The flasks were inoculated with approximately 10 000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator. 

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer at 24, 48, 72, and 96 hours. 

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (1200 µg/L nominal DMBPC concentration) was combined in a 250 mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 48 hours.

Insoluble material was not observed during the test.  The initial measured concentrations ranged from 97 to 108 % of the nominal concentrations and the final measured concentrations were 57 to 76 % of the nominal concentrations.

The algal population grew well, resulting in an average of 4 173 000 cells/mL in the control after 96 hours. No effects were noted during the test. The 0.5 mL aliquot that was incubated for a further 48 hours indicated that any toxic effect at this concentration was algistatic rather than algicidal.

Exposure of algae to DMBPC for 96 hours resulted in a median effective concentration (EC50) that was greater than 1165 µg/L (the highest tested concentration and reported water solubility limit) when calculated using the number of cells/mL or the average specific growth rate, and 1141 µg/L when calculated using the area under the growth curve. The 96 hour no observed effect concentration (NOEC) is 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate, and 161 µg/L when determined using the area under the growth curve.

Description of key information

Under the conditions of this study, the 96 hour ErC50 was > 1165 µg/L. The 96 hour NOEC was 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

595 µg/L

Additional information

The toxicity potential of the test material to freshwater algae was investigated in accordance with the standardised guideline EPA OPPTS 850.5400 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 at 24 ± 2°C with five concentrations of test substance, a control, and a solvent control (0.10 mL/L dimethylformamide). Nominal concentrations were 0 (control and solvent control), 75, 150, 300, 600 and 1200 µg/L. Each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. The flasks were inoculated with approximately 10 000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator. 

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer at 24, 48, 72, and 96 hours. 

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (1200 µg/L nominal DMBPC concentration) was combined in a 250 mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 48 hours.

Insoluble material was not observed during the test.  The initial measured concentrations ranged from 97 to 108 % of the nominal concentrations and the final measured concentrations were 57 to 76 % of the nominal concentrations.

The algal population grew well, resulting in an average of 4 173 000 cells/mL in the control after 96 hours. No effects were noted during the test. The 0.5 mL aliquot that was incubated for a further 48 hours indicated that any toxic effect at this concentration was algistatic rather than algicidal.

Exposure of algae to DMBPC for 96 hours resulted in a median effective concentration (EC50) that was greater than 1165 µg/L (the highest tested concentration and reported water solubility limit) when calculated using the number of cells/mL or the average specific growth rate, and 1141 µg/L when calculated using the area under the growth curve. The 96 hour no observed effect concentration (NOEC) is 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate, and 161 µg/L when determined using the area under the growth curve.