Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

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:

14 November 2017 to 16 October 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98%; Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Yellow Liquid

Analytical monitoring:

yes

Details on sampling:

Aqueous samples (8 mL) were collected from the control and each surviving test concentration at 0 (fresh media, each replicate) and 96 hours (old media, pooled replicates) for analysis. The aqueous samples were diluted with acetonitrile, then diluted further with Elendt M4 medium: acetonitrile, 4:1, v/v as required to bring the response within the calibration range. Duplicate samples were collected, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Vehicle:

no

Details on test solutions:

Preliminary analysis in another study with this UVCB test substance indicated that the test substance was soluble in test media. Test solution preparation followed the method developed in that study. At the start of the test, test substance was weighed onto a glass slide and placed in Elendt M4 medium. The preparation was stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparation was allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 80 mg/L test concentration. The stock solutions were diluted with Elendt M4 media to give the remaining test concentrations. The control solution consisted only of Elendt M4 medium.

The test preparations were observed to be colourless solutions throughout the duration of the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Test organism: Juvenile Daphnia magna (Straus), less than 24 hours old
- Source: Parental laboratory cultures, held and maintained at the test facility (culture was initiated from ephippia obtained from MicroBio Tests Inc., Belgium)
- Age of parental stock: Less than 24 hours old
- Food type: Cultures fed daily with a concentrated suspension of Chlorella vulgaris
- Feeding during test: None

Test type:

static

Water media type:

other: Elendt M4 medium

Limit test:

no

Total exposure duration:

48 h

Hardness:

Not specified

Test temperature:

20 +/- 2 ºC

pH:

7.41 to 7.69

Dissolved oxygen:

8.06 to 9.57 mg/L (85 - 100% air saturation value)

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 0 (control), 5.0, 10, 20, 40 and 80 mg/L
Mean Measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL (nominal) glass, tall form beakers covered with clear lids
- Test solution volume: 50 mL (nominal)
- Aeration: Not specified
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium prepared from trace element stock solutions and reverse-osmosis (RO) water in accordance with OECD Guideline No. 202.

OTHER TEST CONDITIONS
- Intervals of water quality measurement: The pH, dissolved oxygen and temperature were determined in freshly prepared test media at the start of the test and at media renewal at 24 hours and in the old media (pooled replicates) at 24 and 48 hours. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
- Adjustment of pH: None
- Photoperiod: 16-hour light : 8-hour dark
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Statistical analysis was not performed given that no immobilization was observed during the test. The EL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) were estimated empirically by observation of the data.

RANGE-FINDING STUDY
- Nominal test concentrations: Control, 0.010, 0.10, 1.0, 10 and 100 mg/L under static test conditions (duplicate vessels for each)
- Results used to determine the conditions for the definitive study: The definitive test concentrations were based on the results of the range-finding test, which suggested that the 48-hour EC50 value would be between 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

(95% confidence limits, 5.8 – 9.7 mg/L)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

(95% confidence limits, 21 – 27 mg/L)

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Remarks:

(quantitated based on phosphoric acid dimethyl ester constituent)

Basis for effect:

mobility

Details on results:

All daphnids were observed to be submerged in the test solution at 24 and 48 hours.

Results with reference substance (positive control):

- Reference substance: Potassium dichromate
- Test design: Same test organism, medium, exposure conditions and replication as this definitive test
- Results with reference substance valid? Yes; EC50 result was within the expected range state in the OECD Guideline No. 202 (0.6 - 2.1 mg/L).
- Relevant effect levels: 48-hour EC50
- Dose-response test: Yes; treatment concentrations were 0 (control), 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- EC50: 1.366 mg/L
- NOEC: 1.0 mg/L

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The EC50 values were calculated using linear interpolation. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were estimated empirically by observation of the data.

Immobilization in definitive test

Mean Measured Concentration (mg/L)	Number of Daphnia magna exposed	% Immobility at 24 hours	% Immobility at 48 hours
Control	20	-	-
3.9	20	0	5
8.6	20	10	70
20	20	30	100
41	20	100	100
82	20	100	100

 

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated under static conditions and the 48-hour EC50 value was determined to be 6.8 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 3.9 mg/L, based on mean measured concentrations.

Executive summary:

The study was conducted to determine the 48-hour acute toxicity of the test substance against the mobility of the freshwater planktonic crustacean, Daphnia magna according to OECD Guideline No. 202, under GLP conditions. The definitive test was conducted at nominal concentrations of 5.0, 10, 20, 40 and 80 mg/L under static test conditions, with a concurrent control group. At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed for mobility at 24 and 48 hours. The Daphnia magna were not fed during the test.

 

Test solutions were analysed at 0 and 48 hours using high performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) based on quantitation of phosphoric acid dimethyl ester constituent, the marker constituent with the most singularly abundant peak.

 

Analysis of the freshly prepared test media at 0 hours resulted in measured concentrations ranging from 80% to 106% of nominal. Analysis of the old media at 48 hours showed measured concentrations to range from 76% to 100% of nominal value. Given that some of the measured concentrations were outside the 80% to 120% acceptance criteria, and that no clear concentration reduction was observed over the 48-hour period, the mean measured concentrations were calculated and used for effect concentration determination. These were calculated to be 3.9, 8.6, 20, 41 and 82 mg/L (equivalent to 78%, 86%, 100%, 103% and 103% of nominal, respectively).

 

The 48-hour EC50 value was calculated to be 6.8 mg/L. The corresponding NOEC was considered to be 3.9 mg/L.

Description of key information

In an OECD Guideline 202 study, conducted according to GLP, the acute toxicity of the test item to the freshwater invertebrate, Daphnia magna, was investigated under static conditions and the 48-hour EC50 value was determined to be 6.8 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 3.9 mg/L, based on mean measured concentrations (Smithers Viscient (ESG) Ltd., 2018).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.8 mg/L