Orthoboric acid, compound with 2,2',2''-nitrilotriethanol

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:

12 December 2017 to 11 January 2018

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)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples (approximately 150 mL) of the range-finding and definitive test solutions and controls were collected into Teflon® capped glass bottles (250 mL, no preservative).
- Samples were collected immediately prior to test commencement (0 hour) and at test termination (48 hour, from pooled replicates) for analysis.
- All samples were stored at room temperature until transported to ISI and kept refrigerated by ISI until analysis was conducted.
- All samples were analysed within 24 hours of sample collection date.

Vehicle:

no

Details on test solutions:

- Individual test solutions of the test item for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration) prepared in dilution water and without the use of any solubilising agent. All stock and test solutions were corrected for purity of the test material (i.e., 88 % test material, 12 % water).
- Stock solutions were prepared in glass volumetric flasks and stirred until the test material was visibly dissolved using a stir bar and stir plate (approximately 30 and 60 minutes for the range-finding and definitive tests, respectively). The individual test solutions were prepared by adding the appropriate amounts of stock solution and dilution water to achieve the desired test concentrations. The individual test solutions were hand mixed by inverting mixing vessel several times prior to being divided equally into the replicate test vessels. All tests (range-finding and definitive) included a negative control consisting of dilution water with no test material added.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna STRAUS
- Source: The organisms were originally obtained from the Ministry of the Environment and Climate Change (Toronto, Ontario, Canada), the organisms were cultured continuously from a population that has been actively reproducing in the testing laboratory since 1988.
- Age: First instar, neonate (< 24 hours old)
- Feeding during test: no

CULTURE CONDITIONS
- Test organism acclimation was not required, since test organisms were cultured under conditions identical to those of the test.
- Type and amount of food: Once/day: yeast, Cerophyll broth, Trout Chow and Pseudokirchneriella subcapitata algae

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20 ± 2°C

pH:

8.4-8.5

Nominal and measured concentrations:

Nominal: 150 mg/L
Measured: 150.38 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker, 100 mL
- Material, size, headspace, fill volume: 75 mL (min)
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading rate: 15 mL/daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The source of water used for D. magna culturing and testing was reconstituted water prepared following standard operating procedures. A continuous supply of oil-free air was provided to bring the pH and concentrations of dissolved oxygen and other gases into equilibrium with air. The concentration of dissolved oxygen in the water is maintained at > 80 % of the air saturation value.
- Intervals of water quality measurement: Water quality parameters (including dissolved oxygen, pH, and conductivity) were measured in the control and all test solutions at the beginning and end of the test. Temperature was monitored and recorded daily. Water hardness was measured at the beginning of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light, 8-hour dark (with 30 minute transition period)
- Light intensity: 400 to 800 lux (Ambient laboratory illumination)

EFFECT PARAMETERS MEASURED:
- For all test and controls, daphnid immobility was determined after 24 and 48 hours.
- A test organism was considered to be immobile if it was unable to swim within 15 seconds following gentle agitation of the test solution and/or unable to free itself after gentle tapping of the test vessel.

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10, 100 and 300 mg/L
- Concentrations were prepared by adding 0.4, 4, 40 and 120 mL of a nominal 1000 mg/L stock solution, respectively into a 400 mL volumetric flask and making this up to volume with dilution water.
- A total of 10 daphnids (i.e., 2 replicates with 5 daphnids each) were exposed to each test level under static test conditions. All test organisms were transferred to fresh test and the negative control solutions using a pipette and bulb.
- Results used to determine the conditions for the definitive study: Results of the range-finding test indicated that the 48-hour EC50 for daphnid immobilisation was greater than a nominal 300 mg/L concentration of the test material. Therefore, the definitive test was conducted as a Limit Test. To ensure that a measured concentration of 100 mg/L would be maintained during the test, a nominal concentration of 150 mg/L was chosen. The nominal 150 mg/L solution was prepared by adding 150 mL of a 1000 mg/L stock solution of the test material into a 1000 mL graduated cylinder and making this up to volume with dilution water.

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE FINDING TEST
- Confirmation of Exposure Concentrations: Nominal, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 1. With the exception of the lowest (1 mg/L) nominal concentrations, measured (TWM) concentrations were within 10 to 15% of nominal. Additionally, measured concentrations at the beginning and end of the test were within 20% which showed that the test material was stable over the period of the test.
- Immobility: The range-finding test met all the test validity criteria. The percentage immobility of daphnids exposed to the test material during the range-finding test along with the nominal and TWM concentrations are given in Table 2. Due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity. Results of the range-finding test showed that mobility of D. magna was not affected at nominal concentrations of the test material up to 300 mg/L. The test results indicated that the 48-h EC50 for immobility was greater than 300 mg/L nominal concentration (i.e., the highest concentration tested). Based on these results, the definitive test was conducted as a Limit Test at a single concentration of the test material.

DEFINITIVE TEST
- Confirmation of Exposure Concentrations: Nominal, measured and TWM concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. The nominal test material concentration of 150 mg/L gave a TWM concentration of 150.38 mg/L. This TWM concentration was higher than expected based on the results of the range-finding test where initial measured concentrations were approximately 10 to 20% below nominal concentrations, while measured concentration for the definitive test were within 1% of nominal. Reasons for this difference could not be explained as preparation procedures for the stock and test solutions were similar for both tests. The spike recovery was 99.6 to 102.0% which was acceptable.
- Immobility: The definitive tests met all of the test validity criteria. A summary of the test results expressed in terms of the percentage immobility of daphnids observed after 24 and 48 hours is summarised in Table 4. Results of the definitive test showed that there was no (0%) immobility of daphnids exposed to a nominal 150 mg/L solution of the test material. The 48-hour NOEC and LOEC, and the 24 and 48-hour EC50s could not be calculated statistically, since the testing was conducted at a single nominal concentration of the test material. Therefore, 24 and 48-hour EC50s were reported as > 150 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 150 and > 150 mg/L, respectively.

Results with reference substance (positive control):

- Sodium chloride was the reference toxicant used in this study. The LC50 was compared to historical data and was deemed acceptable based on results falling within the 95% confidence interval (warning limit) calculated for the last twenty reference tests.
- In conjunction with the definitive test, the 48-hour LC50 for the Daphnia magna reference toxicant test was 5.6 g/L.

Reported statistics and error estimates:

Statistical analysis of the toxicity test data was not required, since the test was conducted as a Limit test and there was no immobility (0%) of daphnids exposed to either the test material or the controls. Due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, all endpoints from the range-finding and definitive tests were generated using nominal concentrations (corrected for purity).

Table 1: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Range-Finding Test

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 Hours	72 Hours
300	256.36	270.15	263.19
100	89.38d	91.24e	90.31
10	8.30	8.62	8.46
1	0.73	N/A	0.73g
0 (Negative Control)	< MDL	< MDL	< MDL

d Average of duplicate analyses. The Relative Percent Difference (RPD) = 0.4%

e Average of duplicate analyses. RPD = 0.8%

MDL (Method Detection Limit) = 0.2 mg/L

g Based on a single analysis (not a TWM)

N/A = not analysed

 

Table 2: Immobility of D. magna After 24 and 48 Hours Exposure to the Test Material During the Range-Finding Test

Nominal Concentration (mg/L)	Immobility (%)
	T= 24 hours	T= 48 hours
300	0	0
100	0	0
10	0	0
1	0	0
0 (Negative Control)	0	0

 

Table 3: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Definitive Test

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 hrs	48 hrs
150	148.73 and 149.44d	148.52e and 154.88	150.38
0 (Negative control)	< MDL	< MDL	< MDL

d Average of duplicate analyses, Relative Percent Difference (RPD) = 1.3%

e Average of duplicate analyses. RPD = 0.8%

MDL (Method Detection Limit) = 0.2 mg/L

 

Table 4: Immobility of D. magna After 24 and 48 Hours Exposure to the Test Material During the Definitive Test

Nominal Concentration (mg/L)	Immobility out of 20 organisms (%)
	24 h	48 h
150	0	0
0 (Negative control)	0	0

Test Validity Criteria

All of the validity criteria for the definitive tests were met as listed below:

- Immobility of the controls did not exceed 10%.

- Dissolved oxygen concentrations at the end of the test were ≥ 3 mg/L in the control and test solutions.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 24 and 48-hour EC50s based on the results of the definitive test were reported to be > 150 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 150 and > 150 mg/L, respectively.

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions in the daphnia immobilisation test.

Both the range-finding and definitive tests met all the test validity criteria. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity (88% test material; 12% water).

Results of the range-finding test indicated that the 48-h EC50 for immobility was > 300 mg/L. As such, the definitive test was conducted as a Limit Test at a nominal concentration of 150 mg/L.

Under the conditions of this study, the 24 and 48-hour EC50s based on the results of the definitive test were reported to be > 150 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 150 and > 150 mg/L, respectively.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Description of key information

Under the conditions of this study, the 24 and 48-hour EC50s based on the results of the definitive test were reported to be > 150 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 150 and > 150 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

150 mg/L

Additional information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions in the daphnia immobilisation test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Both the range-finding and definitive tests met all the test validity criteria. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity (88% test material; 12% water).

Results of the range-finding test indicated that the 48-h EC50 for immobility was > 300 mg/L. As such, the definitive test was conducted as a Limit Test at a nominal concentration of 150 mg/L.

Under the conditions of this study, the 24 and 48-hour EC50s based on the results of the definitive test were reported to be > 150 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 150 and > 150 mg/L, respectively.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.