Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

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:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples of the test solutions were collected at approx. 0 and 96 hours to measure concentrations of the test substance
- Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers
- At exposure termination, samples were collected from the pooled replicates from each treatment and control group
- All samples were collected in glass vials and processed immediately for analysis
- Samples of freshwater algal medium were fortified at 7.50, 30.0 and 120 mg/L using a stock solution of test item in methanol, and were analyzed concurrently with the test samples
- The measured concentrations for the matrix fortification samples ranged from 90.6 to 102% of fortified concentrations

Vehicle:

no

Details on test solutions:

The test substance used to prepare the test solutions, the analytical matrix fortification samples and the calibration standards was received from Supresta on September 7, 2005. It was assigned Wildlife International, Ltd. identification number 7339 upon receipt and was stored under ambient conditions.

Preparation of Test Concentrations:
- A primary stock solution was prepared by dissolving test item in freshwater algal medium at a nominal concentration of 120 mg/L
- The stock was inverted at least 20 times to mix and appeared clear and colorless
- The primary stock was proportionally diluted with freshwater algal medium to prepare the five additional test solutions at nominal concentrations (7.5, 15, 30, 60 and 120 mg/L)
- All test solutions appeared clear and colorless

Test organisms (species):

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

Details on test organisms:

- Original algal cultures were obtained from the University of Toronto, and had been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland.
- Algal cells used in this test were obtained from Wildlife International, Ltd. cultures that had been actively growing in culture medium for at least
two weeks prior to test initiation.
- The culture was last transferred to fresh medium three days prior to test initiation.
- The negative control organisms were expected to exhibit exponential growth over the 96-hour exposure period.
- Exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve.

Test type:

static

Water media type:

freshwater

Limit test:

no

Hardness:

No data

Test temperature:

23.3-23.9 °C

pH:

7.7-8.5

Dissolved oxygen:

No data

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

Nominal test concentrations of 7.5, 15, 30, 60 and 120 mg/L correspond to mean measured test concentrations of 4.8, 8.3, 15, 33 and 86 mg/L.

Details on test conditions:

I. Culture Medium:
- Culture medium: freshwater algal medium
- Dilution water: purified Wildlife International, Ltd. well water
- pH of the medium: adjusted to 8.0

II. Test Apparatus:
- Test chambers: sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers
- Shaking of the flask: daily, continuously at 100 rpm

III. Environmental Conditions:
- Temperature: 23 ± 2°C, recorded twice daily
- Illumination: 24 hours per day, cool-white fluorescent lighting at an intensity of 6000 ± 20 % lux
- pH-measurement: At test initiation and at exposure termination

IV. Inoculation of Test Chambers:
- Concentration of algal cells in the stock culture: 1.0 x 10 E+6 cells/mL

V. Algal Growth Measurements and Observations:
- Sampling: at approx. 24-hour intervals during the 96-hour exposure
- Storage of samples: held for a maximum of four days under refrigerated conditions
- Cell counter: electronic particle counter (Coulter Electronics, Inc.)
- Primary counting standard: contains Pseudokirchneriella subcapitata cells and the density was verified by a hemacytometer and a microscope

VI. Statistical Analyses:
- Calculation systems: “The SAS System for Windows”, Version 8.02 (5), Microsoft Excel 2000”, TOXSTAT version 3.5

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 86 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence limits not calculable

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 86 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

area under the growth curve, yield

Remarks on result:

other:

Remarks:

95% confidence limits not calculable

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

area under the growth curve, yield

Details on results:

See tables in "Any other information on results incl. tables".

Treatment related effects were observed in the highest test concentrations. Dunnett’s test indicated that cell density, area under the growth curve and growth rate
was significantly reduced (p<0.05) in the 86 mg a.i./L treatment group. Consequently, the 72-hour NOEC for cell density, area under the growth curve and growth rate was 33 mg a.i./L.

Table 1: Measured concentrations of the test item in freshwater algal medium samples

Nominal test concentration (mg/L)	Sample ID (584A-102-)	Sampling time (hours)	Measured concentration* (mg/L) / (mg a.i./L)	Percent of nominal* (%)	Mean measured concentration** (mg/L) / (mg a.i./L)	Mean measured percent of nominal (%)
0	1 7	0 96	<LOQ <LOQ	-- --	--	--
7.5	2 8	0 96	7.31 / 6.18 <LOQ	97.5 --	5.7 / 4.8	76
15	3 9	0 96	15.5 / 13.1 <LOQ	103 --	9.8 / 8.3	65
30	4 10	0 96	30.8 / 26.0 <LOQ	103 --	17 / 15	57
60	5 11	0 96	61.5 / 52.0 15.5 / 13.1	102 25.9	39 / 33	65
120	6 12	0 96	121 / 102 83.8 / 70.8	101 69.9	102 / 86	85

The limit of quantitation (LOQ) was 4.00 mg/L (3.4 mg a.i./L), calculated as the product of the concentration of the lowest calibration standard (4.00 mg/L) and the dilution factor of the matrix blank samples (1.00). 

* Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

** In the absence of a measured concentration at test termination, the LOQ was used in determining the mean measured test concentrations.

 

Table 2: Cell density by replicate over the 96-hour exposure period

Mean measured test concentration (mg a.i./L)	Replicate	Cell density (cells/mL)
		24 hours*	48 hours	72 hours	96 hours
0	A B C	72,475 73,805 84,958	605,923 672,746 653,326	4,074,316 4,543,203 4,708,234	8,188,933 8,137,068 8,683,869
4.8**	A B C	77,322 76,051 76,804	619,525 490,526 547,959	4,593,976 3,697,147 4,271,401	8,191,740 7,304,817 8,528,665
8.3**	A B C	75,158 37,960 77,911	563,090 406,467 374,225	4,521,755 3,835,427 3,325,204	7,475,931 7,138,382 7,947,938
15**	A B C	80,984 79,432 75,302	457,707 480,153 561,093	3,494,836 3,619,623 3,863,582	7,388,814 7,680,114 7,840,309
33	A B C	85,285 87,442 84,260	607,108 414,032 494,878	3,887,603 3,605,507 4,234,823	7,629,965 7,967,982 7,423,131
86	A B C	87,707 80,493 77,552	564,977 452,185 436,743	3,831,371 3,118,994 3,315,377	7,554,859 7,261,375 7,192,431

* The initial cell density of the stock culture was determined and an inoculum volume was administrated to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours).

** In the absence of a measured concentration at test termination, the LOQ (3.4 mg a.i./L) was used in determining the mean measured test concentrations.

 

Table 3: Mean cell density, yield and percent inhibition

Mean measured test concentration (mg a.i./L)	24 hours		48 hours		72 hours		96 hours		96 hours
	Mean cell density ^(1) (cells/mL)	% Inhibition ^(1,2)	Mean cell density ^(1) (cells/mL)	% Inhibition ^(1,2)	Mean cell density ^(1) (cells/mL)	% Inhibition ^(1,2)	Mean cell density ^(1,4) (cells/mL)	% Inhibition ^(1,2)	Mean yield ^(3,4) (cells/mL)	% Inhibition ^(1,2)
0	77,079	--	643,998	--	4,441,918	--	8,336,623	--	8,326,623	--
4.8^(5)	76,726	0.46	552,670	14	4,187,508	5.7	9,568,245	-15	8,000,407	3.9
8.3^(5)	63,676	17	447,927	30	3,894,129	12	7,520,750	9.8	7,510,750	9.8
15^(5)	78,573	-1.9	499,651	22	3,659,347	18	7,363,412	8.4	7,626,412	8.4
33	85,662	-11	505,339	22	3,909,311	12	7,673,693	8.0	7,663,693	8.0
86	81,917	-6.3	484,635	25	3,421,914*	23	7,336,222	12	7,326,222	12

⁽¹⁾ Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

⁽²⁾ Percent inhibition was calculated relative to the negative control replicates.

⁽³⁾ Calculations were performed using Excel 2000. Manual calculations may differ slightly.

⁽⁴⁾ No statistically significant difference (p>0.05) at 72 and 96 hours from the negative control using the Dunnett’s t-test.

⁽⁵⁾ In the absence of a measured concentration at test termination the LOQ (3.4 mg a.i./L) was used in determining the mean measured test concentrations.

* Statistically significant difference (p<0.05) at 72 and 96 hours from the negative control replicates using Dunnett’s test.

 

Table 4: Mean area under the growh curve and percent inhibition

Mean measured test concentration (mg a.i./L)	0-24 hours		0-48 hours		0-72 hours		0-96 hours
	Mean area^(1)	% Inhibition ^(1,2)	Mean area^(1)	% Inhibition ^(1,2)	Mean area^(1)	% Inhibition ^(1,2)	Mean area^(1,3)	% Inhibition ^(1,2)
0	804,952	--	9,217,884	--	70,008,876	--	223,111,368	--
4.8^(4)	800,708	0.53	8,113,456	12	64,755,592	7.5	210,866,576	5.5
8.3^(4)	644,116	20	6,543,360	29	58,408,032	17	195,146,580	13
15^(4)	822,872	-2.2	7,521,556	18	57,189,532	18	192,498,644	14
33	907,948	-13	7,759,968	16	60,495,772	14	199,251,816	11
86	863,008	-7.2	7,421,636	19	54,060,224*	23	182,917,852	18

⁽¹⁾ Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

⁽²⁾ Percent inhibition was calculated relative to the negative control replicates.

⁽³⁾ No statistically significant difference (p>0.05) at 72 and 96 hours from the negative control using the Dunnett’s t-test.

⁽⁴⁾ In the absence of a measured concentration at test termination the LOQ (3.4 mg a.i./L) was used in determining the mean measured test concentrations.

* Statistically significant difference (p<0.05) at 72 and 96 hours from the negative replicates using Dunnett’s test.

 

Table 5: Mean growth rate and percent inhibition

Mean measured test concentration (mg a.i./L)	0-24 hours		0-48 hours		0-72 hours		0-96 hours
	Mean growth rate^(1)	% Inhibition^(1,2)	Mean growth rate^(1)	% Inhibition^(1,2)	Mean growth rate^(1)	% Inhibition^(1,2)	Mean growth rate^(1,3)	% Inhibition^(1,2)
0	0.0850	--	0.0868	--	0.0846	--	0.0701	--
4.8^(4)	0.0849	0.10	0.0835	3.8	0.0838	1.0	0.0696	0.62
8.3^(4)	0.0751	12	0.0789	9.1	0.0827	2.3	0.0690	1.5
15^(4)	0.0859	-1.0	0.0814	6.2	0.0820	3.2	0.0694	1.3
33	0.0895	-5.3	0.0815	6.1	0.0829	2.1	0.0692	1.2
86	0.0876	-3.0	0.0807	7.0	0.0810*	4.3	0.0687	1.9

⁽¹⁾ Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

⁽²⁾ Percent inhibition was calculated relative to the negative control replicates.

⁽³⁾ No statistically significant difference (p>0.05) at 72 and 96 hours from the negative control using the Dunnett’s t-test.

⁽⁴⁾ In the absence of a measured concentration at test termination the LOQ (3.4 mg a.i./L) was used in determining the mean measured test concentrations.

* Statistically significant difference (p<0.05) at 72 and 96 hours from the negative control replicates using Dunnett’s test.

 

Validity criteria fulfilled:

yes

Remarks:

Mean cell density in the control flasks > 16 within 3 days; coefficient of variation of average specific growth rate in the control replicates: < 7 %

Conclusions:

The acute toxicity of diethyl bis(2-hydroxyethyl)aminomethylphosphonate to freshwater algae was investigated in a static GLP study following OECD 201. The 72-hour EC50, based on cell density, area under the growth curve and growth rate, was >86 mg a.i./L, the highest concentration tested. The 96-hour EC50, based on cell density, yield, area under the growth curve and growth rate was >86 mg a.i./L, the highest concentration tested. The 72-hour NOEC, based on cell density, area under the growth curve and growth rate, was 33 mg a.i./L. The 96-hour NOEC, based on cell density, yield, area under the growth curve and growth rate was 86 mg a.i./L, the highest concentration tested. The results relate to measured test concentrations.

Executive summary:

The objective of this study was to determine the toxicity to the freshwater green alga, Pseudokirchneriella subcapitata, over a 96-hour exposure period following the OECD guideline 201. The green alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of five test concentrations and a negative (culture medium) control under static conditions for 96 hours. Three replicate test chambers were maintained in each treatment and control group. Nominal test concentrations were selected in consultation with the Sponsor and were based upon results of an exploratory range finding toxicity test. Nominal test concentration rates selected were 7.5, 15, 30, 60 and 120 mg test substance/L (mg/L) and were corrected to measured test concentrations of 4.8, 8.3, 15, 33 and 86 milligrams active ingredient/L (mg a.i./L) for reporting. Measured test concentrations were determined from samples of test medium collected from each treatment and control group at the beginning and end of the test. At test initiation an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities, which were subsequently used to calculate areas under the growth curve and growth rates. Cell densities, area under the growth curve and growth rates were used to calculate percent inhibition values relative to the control over the 96-hour exposure period. EC50, EbC50 and ErC50 values (i.e., the theoretical toxicant concentrations that would produce a 50% reduction in cell density, area under the growth curve and growth rate, respectively) were calculated, when possible, for each 24-hour interval of the exposure period. In addition, yield was determined at test termination and the EyC50 was calculated at 96-hours. Yield is defined as biomass at the end of the exposure period minus the biomass at the start of the exposure period. No-observed-effect-concentrations (NOECs) were determined at 72 and 96 hours through statistical evaluation of the cell densities, area under the growth curve and growth rates, as well as examination of the concentration-response pattern and observations of the cultures and cell morphology. In addition, the 96 Hour NOEC was based on statistical evaluation of yield. The 72-hour EC50, based on cell density, area under the growth curve and growth rate, was >86 mg a.i./L, the highest concentration tested. The 96-hour EC50, based on cell density, yield, area under the growth curve and growth rate was >86 mg a.i./L, the highest concentration tested. The 72-hour NOEC, based on cell density, area under the growth curve and growth rate, was 33 mg a.i./L. The 96-hour NOEC, based on cell density, yield, area under the growth curve and growth rate was 86 mg a.i./L, the highest concentration tested. The results relate to measured test concentrations.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the Algae study.

.

Description of key information

The acute toxicity of diethyl bis(2-hydroxyethyl)aminomethylphosphonate to freshwater algae was investigated in a static GLP study following OECD TG 201 (Desjardins, 2006). The 72-hour EC50, based on cell density, area under the growth curve and growth rate, was >86 mg a.i./L, the highest concentration tested. The 96-hour EC50, based on cell density, yield, area under the growth curve and growth rate was >86 mg a.i./L, the highest concentration tested. The 72-hour NOEC, based on cell density, area under the growth curve and growth rate, was 33 mg a.i./L. The 96-hour NOEC, based on cell density, yield, area under the growth curve and growth rate was 86 mg a.i./L, the highest concentration tested. The results relate to measured test concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

33 mg/L

Additional information

A fully reliable, guideline, GLP study is available, and therefore considered as key.