Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

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

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Water samples were collected from one test chamber of each treatment and control group one day prior to the start of the test after conditioning the diluter for approximately eight hours
- Samples also were collected from alternating replicate test chambers in each treatment and control group at test initiation and termination to measure concentrations of the test substance
- The samples were collected from mid-depth in each chamber, placed in glass vials, and analyzed as soon as possible without storage

Vehicle:

no

Details on test solutions:

Stock solutions were prepared daily during the test. On each day, one stock solution was prepared for each of the five concentrations tested by mixing calculated amounts of test substance into reverse-osmosis water. The stock solutions were mixed by inversion, and generally ranged in appearance from clear and light brown to clear and brown, increasing in intensity with increasing concentration. The five stock solutions were injected into the diluter mixing chambers (at a rate of 500 µL/minute) where they were mixed with well water (at a rate of 155 mL/minute) to achieve the desired test concentrations of 63, 125, 250, 500 and 1000 mg/L. The test solutions appeared clear and colorless in the diluter mixing chambers and in the test chambers at test initiation and termination.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Origin: obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland
- Growth stage: Juveniles less than 24-hour-old (female)
- Water: Freshwater, moderately-hard water, obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site (USA) (Specific Conductance: 314 µmhos/cm; hardness: 134 mg/L CaCO3; Alkalinity: 180 mg/L CaCO3; pH: 8.2)
- Temperature: 19.8 to 21.7 °C
- Dissolved oxygen concentration (D.O.): 8.2 to 9.1 mg/L ( higher than 91% of saturation),
- pH: 8.3 to 8.8
- Light: Fluorescent light, 16-hour light (800 Iux or less) I 8-hour dark; Light intensity at test initiation was 263 lux
- Culturing: 4 adult daphnids used to supply neonates for the test were held for 21 days prior to collection of the juveniles for testing; adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test
- Feed: daily fed with mixture of yeast, Cerophyll®, and trout chow, as well as a suspension of the freshwater green alga, Selenastrum capricornutum
- Feeding: The adults were fed prior to test initiation, but neonates were not fed during the test

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

132-140 (mg/L as CaC03)

Test temperature:

20 ± 1 °C

pH:

8.1- 8.4

Dissolved oxygen:

8.2 to 9.1 mg/L (>= 91% of saturation)

Salinity:

n.a.

Conductivity:

305-320 (µmhos/cm)

Nominal and measured concentrations:

- Nominal test concentration (mg/L): 63, 125, 250, 500 and 1000
- Mean Measured test concentration (mg/L): 60, 117, 238, 482 and 936. See table 1 in "Any other information on results incl. tables".

Details on test conditions:

I. PREPARATION OF TEST CONCENTRATIONS:
Stock solutions were prepared daily
- On each day, one stock solution was prepared for each of the five concentrations tested by mixing calculated amounts of test substance into reverse-osmosis water
- The stock solutions were mixed by inversion, and generally ranged in appearance from clear and light brown to clear and brown, increasing in intensity with increasing concentration
- The five stock solutions were injected into the diluter mixing chambers (at a rate of 500 µL/minute) where they were mixed with well water (at a rate of 155 mL/minute) to achieve the desired test concentrations
- The test solutions appeared clear and colorless in the diluter mixing chambers and in the test chambers at test initiation and termination

II. EXPERIMENTAL DESIGN:
- Number of loading daphnids: 20 daphnids/test concentration (10 daphnids/vessel)
- Replicate: two
- Test concentrations: 5 (63, 125, 250, 500 and 1000 mg/L)
- Temperature: 20 ± 1 °C
- Light: Fluorescent light, 16-hour light (800 lux or less) I 8-hour dark
- Feeding: None
- Observations: Mortality, immobility and other signs of toxicity were made approximately 7, 24 and 48 hours after test initiation
- Estimation of effective concentraion (EC 50) after 24 and 48 hours: Cumulative percent mortality and immobility observed in the treatment groups were used
- Estimation of no-mortality/immobility concentration and the no-observed-effect concentration (NOEC): determined by visual interpretation of the mortality,
immobility and observation data

III. TEST APPARATUS:
- Apparatus: Continuous-flow diluter
- Pump: Peristaltic pump (Cole-Parmer Instrument Company)

IV. TEST CHAMBER:
- 25 L Teflon®-1ined stainless steel aquaria filled with approximately 22 L of test water
- The depth of the test water in a representative chamber was approximately 28 cm
- Each test chamber contained one test compartment constructed from a glass beaker approximately 6.5 cm in diameter and 12 cm in height, with nylon screen attached to two holes on the sides of the beaker
- Test chambers were indiscriminately positioned in a temperature-controlled water bath set to maintain the desired test temperature
- Each test chamber was labeled with the project number, test concentration and replicate

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 936 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

936 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

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

Table 1: Measured concentrations of the test item in test samples

Nominal test concentration (mg/L)	Sample ID (584A-101-)	Sampling time (hours)	Measured concentration (mg/L)*	Percent of nominal** (%)	Mean measured concentration (mg/L)	Mean measured percent of nominal (%)
0	1 7	0 48	<LOQ <LOQ	-- --	--	--
63	2 8	0 48	59.1 61.7	93.8 98.0	60	95
125	3 9	0 48	114 120	91.4 95.8	117	94
250	4 10	0 48	234 242	93.6 96.8	238	95
500	5 11	0 48	472 492	94.5 98.5	482	96
1000	6 12	0 48	913 958	91.3 95.8	936	94

* The limit of quantitation (LOQ) was 30.0 mg/L, calculated as the product of the concentration of the lowest calibration standard (30.0 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.

 

Table 2: Temperature, dissolved oxygen and pH of water in test chambers

Mean measured concentration (mg/L)	Replicate	0 hours			24 hours		48 hours
		Temp.* (°C)	DO** (mg/L)	pH	DO** (mg/L)	pH	Temp.* (°C)	DO** (mg/L)	pH
0	A B	20.2 20.1	8.6 --	8.2 --	-- 8.3	-- 8.3	20.2 20.2	8.3 --	8.3 --
60	A B	20.1 20.1	8.6 --	8.2 --	-- 7.9	-- 8.2	20.2 20.2	7.4 --	8.2 --
117	A B	20.1 20.1	8.6 --	8.2 --	-- 7.7	-- 8.2	20.2 20.2	7.8 --	8.2 --
238	A B	20.1 20.1	8.4 --	8.1 --	-- 8.2	-- 8.1	20.2 20.2	7.4 --	8.1 --
482	A B	20.2 20.2	8.4 --	8.1 --	-- 8.2	-- 8.1	20.2 20.2	5.7 --	8.0 --
936	A B	20.2 20.2	8.4 --	7.9 --	-- 8.3	-- 7.9	20.2 20.2	7.5 --	8.0 --

* Manual temperature measurements reported. Temperature measured continuously during the test ranged from approximately 20.0 to 20.5°C, measured to the nearest 0.5°C.

** A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20°C in freshwater.

 

Table 3: Cumulative mortality, immobility and observed effects

Mean measured concentration (mg/L)	Replicate	Daphnia /replicate	7 hours			24 hours			48 hours			Percent immobile and dead
			Cumulative dead	Number immobile	Effects*	Cumulative dead	Number immobile	Effects*	Cumulative dead	Number immobile	Effects*
0	A B	10 10	0 0	0 0	AN AN	0 0	0 0	AN AN	0 0	0 0	AN AN	0
60	A B	10 10	0 0	0 0	AN AN	0 0	0 0	AN AN	0 0	0 0	AN AN	0
117	A B	10 10	0 0	0 0	AN AN	0 0	0 0	AN AN	0 0	0 0	AN AN	0
238	A B	10 10	0 0	0 0	AN AN	0 0	0 0	AN AN	0 0	0 0	AN AN	0
482	A B	10 10	0 0	0 0	AN AN	0 0	0 0	AN AN	0 0	0 0	AN AN	0
936	A B	10 10	0 0	0 0	AN AN	0 0	0 0	AN AN	0 0	0 0	AN AN	0

* Observed effects: AN = appear normal.

Validity criteria fulfilled:

yes

Remarks:

(The immobilisation and other abnormalities in the controls did not exceed 10 % by the end of the test. The dissolved oxygen concentration remained above 3 mg/L throughout the exposure period)

Conclusions:

The acute toxicity of diethyl bis(2-hydroxyethyl)aminomethylphosphonate to daphnia magna was investigated in a flow-through GLP study following OECD 202. The study reported of no effects after 48 hours of exposure. An EC50 of higher than 936 mg/L (arimethic mean of the analytically determined test concentrations) was determined. The no-mortality/immobility concentration and the NOEC were 936 mg/L.

Executive summary:

The acute toxicity of diethyl bis(2-hydroxyethyl)aminomethylphosphonate to daphnia magna was investigated in a flow-through GLP study following OECD 202. Daphnids were exposed to a geometric series of five test concentrations and a negative (dilution water) control for 48 hours under flow-through conditions. Two replicate test chambers were maintained in each treatment and control group, with 10 daphnids in each test chamber for a total of 20 daphnids per concentration. Nominal test concentrations were selected in consultation with the Sponsor based on the results of an exploratory range finding toxicity test. Nominal test concentrations selected were 63, 125, 250, 500 and 1000 mg test item. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning and end of the test. Delivery of the test substance was initiated one day prior to test initiation in order to achieve equilibrium of the test substance in the test chambers. The concentration in the test chambers was confirmed prior to the start of the test after conditioning the diluter for approximately eight hours. Daphnids were impartially assigned to test chambers at test initiation. Observations of mortality, immobility and other signs of toxicity were made approximately 7, 24 and 48 hours after test initiation. Cumulative percent mortality and immobility observed in the treatment groups were used to estimate EC50 values at 24 and 48 hours. The no-mortality/immobility concentration and the noobserved-effect concentration (NOEC) were determined by visual interpretation of the mortality, immobility and observation data. An EC50 of higher than 936 mg/L (arimethic mean of the analytically determined test concentrations) was determined. The no-mortality/immobility concentration and the NOEC were 936 mg/L. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute Daphnia study.

Description of key information

The acute toxicity of diethyl bis(2-hydroxyethyl)aminomethylphosphonate to Daphnia magna was investigated in a flow-through GLP study following OECD TG 202 (Palmer, 2005). The study reported of no effects after 48 hours of exposure. An EC50 of higher than 936 mg/L (arimethic mean of the analytically determined test concentrations) was determined, corresponding to the highest tested concentration. The no-mortality/immobility concentration and the NOEC were 936 mg/L.

Additional information

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