Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- and Guideline compliant study, no deviations to guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 1000 mg test item/L
- Sampling method: Samples of media for chemical analysis were taken from the preparation vessels at the start of the test period. At the end of the 48 h test period samples were taken from the test vessels by combining the contents of the replicates. 100 mL portions were collected and analysed for TOC
- Sample storage conditions before analysis: The remaining media were stored at 4°C. Samples were analysed within 2 days after sampling.

Vehicle:

no

Details on test solutions:

Methyl borate esters is a mixture containing components that are soluble in water at the highest nominal test concentration. The test solutions were therefore prepared by direct addition to the appropriate test medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea, Daphnia magna
- Strain: Daphnia magna (Straus) culture is derived from individuals obtained from Zeneca Brixham Laboratory. The Zeneca culture originates from a clone obtained from I.R.Ch.A. France.
- Source: The culture is derived from individuals obtained from Zeneca Brixham Laboratory. The Zeneca culture originates from a clone obtained from I.R.Ch.A. France.
- Age at study initiation (mean and range, SD): < 24 h
- Weight at study initiation (mean and range, SD): NA
- Length at study initiation (length definition, mean, range and SD): NA
- Valve height at study initiation, for shell deposition study (mean and range, SD): NA
- Peripheral shell growth removed prior to test initiation: NA
- Method of breeding: For the first 14 days the D. magna are cultured in 1 litre pyrex glass beakers containing 0.8 Llitre of reconstituted freshwater. From day 15 onwards, they are cultured in a 2 litre beaker containing 1.5 litre of reconstituted freshwater. Each vessel and its contents are referred to as a "culture". New cultures are started with animals less than 24 hours old, at a density of about 10 per vessel. The cultures are held in a temperature controlled room, normally 18-22°C, under artificial light in a 16 h light 8 h dark cycle. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris to give a concentration of aproximately 100.000 cells /mL. The C. vulgaris is obtained from a 4 litre culture grown under semi-axenic conditions. Prior to the use the C. vulgaris is concentrated by filtration and resuspension in sterilized C. vulgaris growth medium. The C. vulgaris is stored in a refrigerator at 4°C and used within one week.
- Feeding during test: None

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

See Table 1

Test temperature:

See Table 1

pH:

See Table 1

Dissolved oxygen:

See Table 1

Nominal and measured concentrations:

See Table 2

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL crystallizing dishes were filled with 100 mL test solution / reconstituted fresh water.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): NA
- Biomass loading rate: 0.1 daphnia per mL


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing of D. magna is a reconstituted fresh water prepared by dissolving the following amounts of Analar grade salts in reverse osmosis water:
NaHCO3: 192 mg/L
CaSO4*2H2O: 120 mg/L
MgSO4*7H2O: 240 mg/L
KCL: 8 mg/L
The recipe has been recommended as one suitable for producing a "hard" water by the U.S. EPA (1975); (U.S. Environmental Protection Agency 1975; Methods for acute toxicity testing with fish, macroinvertebrates and amphibians. EPA-660/3-75-009.)
- Culture medium different from test medium: The culture medium received 20 mL soil extract per L.
- Intervals of water quality measurements:
Temperature: continuous water temperature measurements in one test vessel (1 min intervals)
The pH and concentration of dissolved oxygen were determined in each medium at the start of the test and in a control test vessel and one test vessel containing each medium at the end of the test. The total hardness of the control medium was determined at the start of the test.


OTHER TEST CONDITIONS
- Adjustment of pH:Not stated
- Photoperiod: only stated for the culture: 16 h light 8 hours dark
- Light intensity: not stated


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization at 24 and 48 hours after start of the exposure.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0 (control) and nominal 1000 mg methyl borate esters/L (mean measured concentration 960 mg methyl borate esters/L)
- Justification for using less concentrations than requested by guideline: a limit test is foreseen in the guideline, based on results of a range-finding study
- Range finding study
- Results used to determine the conditions for the definitive study: The 48-hour EC50 as determined in a range finding test was > 1000 mg methyl borate esters/L. Further results were not reported.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

< 211.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

< 960 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 211.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 960 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 211.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 960 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 211.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 960 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

converted from TOC

Basis for effect:

other: Immobilization

Details on results:

- Behavioural abnormalities: none reported
- Mortality of control: no immobilization in the control
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: no

4 out of 20 daphnids were immobilized in the measured 960 mg/L group. Therefore, the EC50 is > 960 mg methyl borate esters/L and the NOEC is < 960 mg methyl borate esters/L

Reported statistics and error estimates:

Since the test was performed as limit test, no statistical analysis was performed.
Since only 20% of the exposed daphinds were immobilized, the EC50 was reported as > 960 mg methyl borate ester /L (mean measured) and the NOEC was reported as < 960 mg methyl borate ester /L (mean measured) .

Table 1 Water quality during the test

Parameter	Target	Actual
Temperature [°C]	18-22	20 – 20.8
pH	Change < 1	8.0- 8.6
Dissolved Oxygen [mg/L]	>= 5.5	9.1 – 9.2
Total hardness as CaCO3 [mg/L]	-	166

 

Table 2 Measured concentrations of methyl borate esters in the test medium

Nominal concentration	Measured concentration of methyl borate esters in test media as TOC (mg/L)and also converted to mg/L of methyl borate esters*
	0 h	48 h
Control	< 10 mg TOC /L	< 10 mg TOC /L
	< 20 mg methyl borate esters /L	< 20 mg methyl borate esters /L
1000 mg/L	510 mg TOC /L	490 mg TOC /L
	970mg methyl borate esters /L	940   mg methyl borate esters /L

*  Conversation from TOC (in mg/L) to mg methyl borate esters/L employs an analytical determined conversation factor of 1.92.

 

Table 3 Immobilisation of D. magna during the study

Nominal concentration of methyl borate ester (mg/L)	Replicate	Number of Daphnia per replicate	Number of immobilized Daphina		TOTAL immobilised
			24 h	48 h
Control	A	10	0	0	0
	B	10	0	0
1000	A	10	3	3	4 (20%)
	B	10	1	1

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50 is > 960 mg methyl borate esters/L (mean measured), corresponding to >211.2 mg B-TEGME/L.

Executive summary:

The acute toxicity of methyl borate esters (which contains 22% B-TEGME) to Daphnia magna was determined in a 48 h GLP compliant toxicity limit test without renewal of the test media. The test was performed according to OECD 202. The nominal test concentrations were 0 (control) and 1000 mg methyl borate esters/L, corresponding to 0 (control) and 220 mg B-TEGME/L, respectively. The mean measured concentration was 960 mg methyl borate esters/L corresponding to 211.2 mg B-TEGME/L. Within the first 24 hoursof exposure, 0 and 4 out of 20 daphnids were immobilized in the control and the mean measured 960 mg methyl borate esters/L test group, respectively. Thereafter, no further immobilization was observed. The 24 and 48 -hour EC50 were both > 960 mg methyl borate esters/L (mean measured). This corresponds to >211.2 mg B-TEGME/L.

 

Methyl borate esters is a mixture containing components that are soluble in water, therefore the test media were produced by direct addition of the test substance to the appropriate media. The concentrations of methyl borate ester were monitored at the start and termination of the study using TOC analysis. A factor to convert TOC results to methyl borate esters concentrations was employed. The measured concentrations were in agreement with the nominal concentrations. The test results are expressed as arithmetic means of the measured concentrations of methyl borate esters in the test solutions.

The effect concentrations for B-TEGME were calculated based on the the presence of 22% B-TEGME in methyl borate esters.

Description of key information

 EC50 > 211.2 mg/L acute Daphnia magna immobilization (OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

211.2 mg/L

Additional information

For the assessment of the EC50 for freshwater invertebrates, three acute Daphnia studies are available, one key study with B-TEGME (BASF 1988), one key study with a mixture of methyl borate esters which contains 22% B-TEGME (Shell 1999b) and one supporting study with a brake fluid containing 17% B-TEGME (Shell 1987b).

In the non-GLP (key) study from BASF (1988) the acute toxicity of B-TEGME to Daphnia magna was determined in a 48 hour static study. The study was performed according to the EU C.2 test guideline. Restrictions of this study are: (1) the study was conducted non-GLP, (2) the purity of B-TEGME is not provided, (3) dose verification analysis was not performed. The advantage of this study is that the study was performed with B-TEGME as test item itself and not with a B-TEGME containing mixture. The nominal test concentrations were 0 (control), 31.25, 62.5, 125, 250 and 500 mg B-TEGME/L. The control and each test group was started with 4 replicates (5 daphnids each). At start of the exposure, the daphnids were younger than 24 hours. Within 48 hours of exposure, none of the daphnids from the control and treated test groups was immobilized. The 24 and 48-hour EC0 was 500 mg B-TEGME/L and the EC10, EC50 and EC100 were all > 500 mg B-TEGME/L.

In the GLP compliant (key) study from Shell (1999) the acute toxicity of methyl borate esters (containg 22% B-TEGME) to Daphnia magna was determined in a static 48 h toxicity limit test. The test was performed according to OECD 202. No relevant restrictions appeared and the only disadvantage of this study is the fact that the study was performed with a B-TEGME containing mixture and not with B-TEGME as pure substance itself. The nominal test concentrations were 0 (control) and 1000 mg methyl borate esters/L, corresponding to 0 (control) and 220 mg B-TEGME/L. The mean measured concentration was 960 mg methyl borate esters/L, corresponding to 211.2 mg B-TEGME/L. The control and the treated test group was started with 2 replicates (10 daphnids each). Within the first 24 hours of exposure, 0 and 4 out of 20 daphnids were immobilized in the control and at 211.2 mg B-TEGME/L, respectively. Thereafter, no further immobilization was observed. The 24 and 48 -hour EC50 were both >211.2 mg B-TEGME/L.

In the GLP compliant (supporting) study from Shell (1987b) the acute toxicity of a brake fluid (which contains 17% B-TEGME) to Daphnia magna was determined in a static 48 h toxicity test. The test design was similar to OECD 202. Restrictions of this study are (1) no specific guideline was followed (2) dose verification analysis was not performed (3) the temperature of 18- 22°C exceeded the maximum allowable variation of +/- 1°C as given in the OECD 202 test guideline. Furthermore, the test substance was a mixture of B-TEGME and other components. The nominal test concentrations were 0 (control), 3.4, 8.5, 17, 34, 85, and 170 mg B-TEGME/L, applied as 0 (control), 20, 50, 100, 200, 500, and 1000 mg brake fluid /L. For the control and each of the treated test groups three replicates with 10 daphnids younger than 24 hours were set up. Within the 48 hour exposure period, 1 out of 30 daphnids was immobilized in the 17, 34 and 170 mg B-TEGME/L test groups. The 24 and 48 -hour EC50 values were both >170 mg B-TEGME/L.

 

The key study of BASF (1988) and Shell (1999) shows EC50 of > 500 mg B-TEGME/L and >211.2 mg B-TEGME/L, respectively. Due to the above given restrictions, the EC50 of BASF is considered less reliable than the EC50 value of Shell (1999). Moreover, the EC50 of Shell (1999) is more conservative than the EC50 of BASF. The supporting study of Shell (1987) provides an EC50 value of > 170 mg B-TEGME/L. Due to the above given restrictions, this value is considered less reliable than the value provided by Shell (1999).

In conclusion, the EC50 value of 211.2 mg B-TEGME/L is considered reliable and sufficiently conservative to assess the toxicity of B-TEGME on aquatic invertebrates.

The above value is higher than the limit for classification and labeling, i. e., higher than 100 mg B-TEGME/L.