bis(diethylamino)-N,N-diethylmethaniminium chloride

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

Study period:

January 7, 2004 to April 20, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP

Analytical monitoring:

yes

Details on sampling:

Analytical determination of test substance concentration (active ingredient) was performed with samples collected from stock solutions prepared at each concentration prior to their distribution to test vessels at the start of the toxicity test and from each replicate test vessel after 72 hours. Samples collected from replicate test vessels at the end of the test were pooled.

Vehicle:

no

Details on test solutions:

The dilution water used for acclimation of test organisms and for all toxicity testing was sterile freshwater AAP medium at a pH of 7.5 ± 0.1 (the pH was adjusted with 0.1N NaOH). A sample of the dilution water used for this test contained 1.3 mg/L total organic carbon.

A stock solution with a nominal concentration of 800 mg a.i./L was prepared (with correction for purity) by bringing 1.2575 g of test substance to a total volume of 1,000 mL with algal medium in a glass volumetric flask. This stock solution was used to prepare 13, 25, 50, 100, 200, 400, and 800 mg a.i./L test solutions by transferring the appropriate amount of stock solution into algal medium (the control was 400 mL of algal medium). The pH of the solutions was adjusted to 7.5 ± 0.1. A stability blank sample was set up at 800 mg a.i./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) were from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin. 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 prior to the start of the test, the temperature ranged from 23.6 to 24.7 °C and the light intensity was 50 to 51 microEin/m²sec (24 hours light/0 hours dark photoperiod).
During the acclimation period, the culture was actively growing in at least two 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 four 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:

72 h

Test temperature:

23.4 to 23.7 °C

pH:

7.5 to 7.6 (initial); 7.2 to 9.3 (termination)

Nominal and measured concentrations:

Nominal: 0 mg a.i./L (control), 13, 25, 50, 100, 200, 400 and 800 mg/L
Mean Measured: ND (none detected, control), 12, 23, 47, 95, 190, 380 and 790 mg a.i./L

Details on test conditions:

A range-finding test was conducted with a control and five concentrations of the test substance: 5.0, 10, 50, 100, and 500 mg a.i./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: 5.0 and 10 mg a.i./L = greater than 95%, 50 mg a.i./L = 35%, 100 mg a.i./L = 14%, and 500 mg a.i./L = 9%. Insoluble material was not observed. A definitive test was conducted with a control and five concentrations of the test substance: 13, 25, 50, 100, and 200 mg a.i./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: 13 mg a.i./L = 91%, 25 mg a.i./L = 70%, 50 mg a.i./L = 52%, 100 mg a.i./L = 35%, and 200 mg a.i./L = 14%. Insoluble material was not observed. The test was repeated with higher concentrations to allow EC50 calculations using the average specific growth rate.

The final definitive toxicity test was conducted at 24 ± 2°C with seven concentrations of test substance and a control. Water quality measurements were made and each solution was subdivided into clear glass 250 mL Erlenmeyer flasks (three replicates of the control and each test concentration). Flasks were inoculated with approximately 10,000 algal cells/mL and capped with clear glass inverted 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) and repositioned by five places each day. A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool white fluorescent lights that provided a light intensity of approximately 370 to 390 footcandles (also measured as approximately 51 to 53 microEin/m²sec).

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer at 24, 48, and 72 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 with noted interruption(s). 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.

At the conclusion of the toxicity test, a determination of whether the toxic effects observed at 95, 190, 380, and 790 mg a.i./L were algistatic or algicidal was made by transferring 0.50 mL of test solution from each flask to 100 mL of dilution water without the test substance and incubating these flasks under test conditions for 96 to 120 hours. At the conclusion of this incubation period, the number of cells/mL was determined in each flask.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

620 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 500-780 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

280 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 220-370 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

180 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 140-250 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

47 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Insoluble material was not observed during the definitive toxicity test. The mean measured concentrations were 92 to 99% of nominal concentrations, indicating that the test substance was stable under test conditions. All results were based on mean measured test concentrations. The algal population grew well, resulting in an average of 768,000 cells/mL in the control after 72 hours. No effects (relative size differences, unusual cell shapes, colors, 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 23.4 to 23.7°C.

At the conclusion of the final definitive toxicity test, a 0.5 mL aliquot of test media from the 95, 190, 380, and 790 mg a.i./L concentrations was combined with 100 mL of fresh media and incubated under test conditions. During a 96-hour incubation period, the algal population increased from approximately 3,500 cells/mL to 726,000 cells/mL at 95 mg a.i./L and from approximately 890 cells/mL to 206,000 cells/mL at 190 mg a.i./L. During a 120-hour incubation period, the algal population grew from approximately 620 cells/mL to 214,000 cells/mL at 380 mg a.i./L and from approximately 470 cells/mL to 154,000 cells/mL at 790 mg a.i./L, indicating that the toxic effects were 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 24, 48, and 72 hour EC50 values were calculated using a weighted least squares non-linear regression technique, when possible. The slope of the concentration-response curve is not calculated by this method. The no observed effect concentration (NOEC) was determined using a one-way analysis of variance (ANOVA) and Bonferroni’s test. The NOECs were determined using the mean measured concentrations of the test substance, and the number of cells/mL and average specific growth rate. Treatment data were compared to control data for the determination of the NOECs.

Measured concentrations of the test substance during the toxicity test with the freshwater alga, Selenastrum capricornutum.

Nominal Concentration of the Test Substance     (mg a.i./L)	Measured Concentrations of the Test Substance (mg a.i./L)
	0 Hour	72 Hour	Mean	% Recovery
Test Media Samples
Control	ND	ND	ND	--
13	12	11	12	92
25	23	23	23	92
50	47	47	47	94
100	95	94	95	95
200	190	190	190	95
400	380	380	380	95
800	800	770	790	99
Stability Blank
800	800	820	810	101
Matrix Spike Blank
100	--	100	--	--
	--	100	100	100
Laboratory Control Spike Sample
100	99	99	99	99
Blank
0	ND	ND	ND	--
ND = none detected at or above the limit of quantitation

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

Mean Measured Conc. of the Test Substance (mg a.i./L)	R	Number of Cells per Milliliter
		Hour of Exposure
		0	24	48	72
Control	1	10,000	46,000	200,000	802,000
	2	10,000	49,000	246,000	740,000
	3	10,000	48,000	216,000	762,000
	Mean	10,000	48,000	221,000	768,000
12	1	10,000	56,000	236,000	820,000
	2	10,000	62,000	206,000	766,000
	3	10,000	60,000	210,000	810,000
	Mean	10,000	59,000	217,000	799,000
	% control	100	123	98	104
23	1	10,000	48,000	144,000	630,000
	2	10,000	58,000	168,000	672,000
	3	10,000	66,000	188,000	684,000
	Mean	10,000	57,000	167,000	662,000
	% control	100	119	76	86
47	1	10,000	39,000	166,000	502,000
	2	10,000	50,000	146,000	676,000
	3	10,000	47,000	150,000	612,000
	Mean	10,000	45,000	154,000	597,000
	% control	100	94	70	78
95	1	10,000	45,000	90,000	192,000
	2	10,000	50,000	120,000	260,000
	3	10,000	48,000	118,000	238,000
	Mean	10,000	48,000	109,000	230,000
	% control	100	100	49	30
190	1	10,000	50,000	54,000	54,000
	2	10,000	33,000	62,000	58,000
	3	10,000	46,000	60,000	66,000
	Mean	10,000	43,000	59,000	59,000
	% control	100	90	27	8
380	1	10,000	21,000	39,000	40,000
	2	10,000	42,000	32,000	34,000
	3	10,000	33,000	38,000	48,000
	Mean	10,000	32,000	36,000	41,000
	% control	100	67	16	5
790	1	10,000	20,000	17,000	31,000
	2	10,000	21,000	34,000	27,000
	3	10,000	18,000	28,000	35,000
	Mean	10,000	20,000	26,000	31,000
	% control	100	42	12	4

R= replicate

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

Mean Measured Conc. of the Test Substance (mg a.i./L)		Average Specific Growth Rate
		Hours of Exposure
		24	48	72
Control	Mean	0.065	0.064	0.060
12	Mean	0.074	0.064	0.061
	% control	114	100	102
23	Mean	0.073	0.059	0.058
	% control	112	92	97
47	Mean	0.063	0.057	0.057
	% control	97	89	95
95	Mean	0.065	0.050	0.044
	% control	100	78	73
190	Mean	0.061	0.037	0.025
	% control	94	58	42
380	Mean	0.048	0.027	0.020
	% control	74	42	33
790	Mean	0.029	0.020	0.016
	% control	45	31	27

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study exposure of algae to to the test material for 72 hours resulted in an EC50 of 58 mg/L (95 % confidence interval = 40 to 85 mg/L) when determined using the number of cells/mL and 180 mg/L (95 % confidence interval = 140 to 250 mg/L) when determined using the average specific growth rate. When treatment data are compared to the control, the 72 hour NOEC is 12 mg/L test material when determined using the number of cells/mL and 47 mg/L when determined using the average specific growth rate.

Executive summary:

The acute toxicity of the test material to freshwater algae, Selenastrum capricornutum, was investigated according to the standardised guidelines OECD 201, under GLP conditions. 

The test was performed under static conditions with seven concentrations of test material and a dilution water control at 24 ± 2 °C.  Nominal concentrations of the test material were 0 mg/L (control), 13, 25, 50, 100, 200, 400, and 800 mg/L.  Mean measured concentrations were ND (none detected; control), 12, 23, 47, 95, 190, 380, and 790 mg/L. The mean measured concentrations were 92 to 99 % of nominal concentrations, indicating that the test material was stable under test conditions.  Insoluble material was not observed at any tested concentration during the test.

The dilution water was sterile freshwater AAP medium adjusted to a pH of 7.5 ± 0.1.  The test was performed in clear glass 250 mL flasks that contained 100 mL of test solution.  Algae used in the test were acclimated to test conditions for more than 14 days.  Algae were distributed among three replicates of the control and each test concentration at the rate of approximately 10,000 cells/mL.

Under the conditions of this study exposure of algae to to the test material for 72 hours resulted in an EC50 of 58 mg/L (95 % confidence interval = 40 to 85 mg/L) when determined using the number of cells/mL and 180 mg/L (95 % confidence interval = 140 to 250 mg/L) when determined using the average specific growth rate. When treatment data are compared to the control, the 72 hour NOEC is 12 mg/L test material when determined using the number of cells/mL and 47 mg/L when determined using the average specific growth rate.

Description of key information

Ward, Wyskiel, Boeri (2003)

Under the conditions of this study exposure of algae to to the test material for 72 hours resulted in an EC50 of 58 mg/L (95 % confidence interval = 40 to 85 mg/L) when determined using the number of cells/mL and 180 mg/L (95 % confidence interval = 140 to 250 mg/L) when determined using the average specific growth rate. When treatment data are compared to the control, the 72 hour NOEC is 12 mg/L test material when determined using the number of cells/mL and 47 mg/L when determined using the average specific growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

180 mg/L

EC10 or NOEC for freshwater algae:

47 mg/L

Additional information

Ward, Wyskiel, Boeri (2003)

The acute toxicity of the test material to freshwater algae, Selenastrum capricornutum, was investigated according to the standardised guidelines OECD 201, 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 test was performed under static conditions with seven concentrations of test material and a dilution water control at 24 ± 2 °C.  Nominal concentrations of the test material were 0 mg/L (control), 13, 25, 50, 100, 200, 400, and 800 mg/L.  Mean measured concentrations were ND (none detected; control), 12, 23, 47, 95, 190, 380, and 790 mg/L. The mean measured concentrations were 92 to 99 % of nominal concentrations, indicating that the test material was stable under test conditions.  Insoluble material was not observed at any tested concentration during the test.

The dilution water was sterile freshwater AAP medium adjusted to a pH of 7.5 ± 0.1.  The test was performed in clear glass 250 mL flasks that contained 100 mL of test solution.  Algae used in the test were acclimated to test conditions for more than 14 days.  Algae were distributed among three replicates of the control and each test concentration at the rate of approximately 10,000 cells/mL.

Under the conditions of this study exposure of algae to to the test material for 72 hours resulted in an EC50 of 58 mg/L (95 % confidence interval = 40 to 85 mg/L) when determined using the number of cells/mL and 180 mg/L (95 % confidence interval = 140 to 250 mg/L) when determined using the average specific growth rate. When treatment data are compared to the control, the 72 hour NOEC is 12 mg/L test material when determined using the number of cells/mL and 47 mg/L when determined using the average specific growth rate.