BAP

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:

6 February 2018 to 27 February 2019

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: >95% (This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB))
Description: Brown paste to very viscous liquid

Analytical monitoring:

yes

Details on sampling:

At the start of the test (0 hours) and at media renewal at 24 hours, 10 mL samples of freshly prepared test media were taken from the control and the test media preparation vessel for chemical analysis. At 24 and 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media from the test and control groups. In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.

Concentrations of the test substance were determined by diluting aqueous samples with acetonitrile containing 0.2% formic acid, then diluting further with Elendt M4 medium: acetonitrile (1:1, v/v) containing 0.1% formic acid as required to bring the response within the calibration range.

Vehicle:

no

Details on test solutions:

The test material is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with anticipated low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guidance, the test material was prepared as Water Accommodated Fractions (WAFs).

At the start of the test and media renewal at 24 hours, ca 1.0 mg of test substance was weighed onto a glass slide and placed in 1000 mL of 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 1.0 mg/L loading rate WAF. Dilutions were prepared from the 1.0 mg/L loading rate WAF to give the test series. A control group was prepared in the same manner without the addition of test substance.

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:

semi-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:

7.81 - 8.02

Dissolved oxygen:

8.57 - 9.72 mg/L (96-102% air saturation value, ASV)

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

The nominal concentrations tested were 0 (control), 0.019, 0.041, 0.091, 0.20, 0.45 and 1.0 mg/L.

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 aerated during the test
- 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: At the start of the test (0 hours) and at media renewal at 24 hours, 10 mL samples of freshly prepared test media were taken from the control and the test media preparation vessel for chemical analysis.
At 24 and 48 hours, 10 mL samples were also taken for chemical analysis from the pooled expired test media from the test and control groups.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
- Adjustment of pH: None
- Photoperiod: 16-hour light : 8-hour dark
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
After 24 and 48 hours, the Daphnia magna in each test vessel were observed for evidence of immobility. The EL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) at 24 and 48 hours were determined, if possible.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

/ loading rate WAF

Basis for effect:

mobility

Results with reference substance (positive control):

A positive control study was conducted to determine the 48-hour acute toxicity of the reference substance, potassium dichromate, on the freshwater planktonic crustacean, Daphnia magna. The 48-hour EC50 values for mobility was determined by linear interpolation to be 1.366 mg/L. The 48-hour NOEC value was determined to be 1.0 mg/L. These results were within the expected toxicity range (48-hour EC50: 0.6 - 2.1 mg/L) for this reference substance and all validity criteria were met.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The EL50 values at 24 and 48 hours and the no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) at 24 hours were established by empirical evaluation of the data. The NOEL and LOEL values at 48 hours were determined using Fisher Exact/Bonferroni-Holm Test.

The results of the range-finding test conducted under static conditions suggested that the EL₅₀may be less than 0.10 mg/L loading rate WAF. The temperature during the range-finding tests differed by more than 1ºC from initial measured temperatures which deviated from the protocol. This deviation was considered not to impact on the integrity of the test given that no immobilisation was observed and temperatures remained within the 20 ± 2ºC range specified in the protocol.

Initial Definitive Test

Immobilisation was not observed at any of the loading rates tested. Chemical analysis of the freshly prepared test media at 0 and 24 hours showed measured concentrations to range from less than the limit of quantification (LOQ) of the analytical method (assessed as 0.001 mg/L) to 0.00748 mg/L. Analysis of the corresponding old media samples at 24 and 48 hours showed measured concentrations be <LOQ.

Analysis of the 1.0 mg/L loading rate WAF, which was used to prepare the test solutions, at 0 and 24 hours showed measured concentrations of 0.327 and 0.0948 mg/L, respectively.

The analytical batch comprised of the 0-hour samples failed the acceptability criteria due to poor calibration line and recoveries; these results should therefore be evaluated with caution.

Final Definitive Test

Chemical Analysis

The limit of quantification (LOQ) was 0.0010 mg/L.

Analysis of the test media samples was conducted at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media).

Chemical analysis of the freshly prepared test media at 0 and 24 hours showed measured concentrations to range from less than the limit of quantification (LOQ, 0.001 mg/L) of the analytical method to 0.0790 mg/L. Analytical results for several of the freshly prepared WAF solutions at 24 hours (0.041 through 1.0 mg/L LR WAF) were outside the calibration range and these extrapolated values therefore should be evaluated with caution.

Analysis of the corresponding old media samples at 24 and 48 hours showed measured concentrations to range from <LOQ to 0.0173 mg/L.

The analytical results from the four tests showed considerable variation. The test substance is a complex UVCB substance. As such the test substance was prepared using a WAF method of preparation. Whilst ever effort was employed to ensure that the WAFs were prepared in identical manners, the WAFs were removed by siphon without filtration and small variations in solution preparation (e.g., stirring speed) could result in variable solubility of the marker component or the formation of micelles which would not be visible. The presence of micelles can result in an apparent increase in measured concentration.

The numbers of immobilised Daphnia magna following exposure to the test substance for 24 and 48 hours are presented below:

Nominal loading rate (mg/L)	Number of Daphnia magna exposed	% Immobility at 24 hours	% Immobility at 48 hours
Control	20	0	0
0.019	20	0	0
0.041	20	5	5
0.091	20	5	5
0.20	20	0	10
0.45	20	0	5
1.0	20	10	10

Statistical analysis showed the observed immobilisation was not significant compared to the control.

Validity criteria fulfilled:

yes

Remarks:

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied.

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated under semi-static conditions and the 48-hour EL50 value was determined to be >1.0 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 1.0 mg/L, based on loading rate WAF.

Executive summary:

The objective of the study was to determine the 48-hour acute toxicity of the test material against the mobility of the freshwater planktonic crustacean, Daphnia magna.

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

The test substance, phosphoric acid, butyl ester, compds. with branched dodecylbenzenamine, is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with anticipated low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test substance was prepared as Water Accommodated Fractions (WAFs).

Based on the results of two range-finding tests and an initial definitive test, for which only the key findings have been reported, the final definitive test was conducted at nominal loading rates of 0.019, 0.041, 0.091, 0.20, 0.45 and 1.0 mg/L using semi-static test conditions. A concurrent control group was also included.

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 at 24 and 48 hours. The Daphnia magna were not fed during the test.

Samples of test media were analysed at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media). Concentrations of the test substance were determined by liquid chromatography triple quadrupole mass spectrometry (LC-TQMS) system, using the branched dodecylbenzene amine constituent as an analytical marker for quantification.

Analysis of the freshly prepared test media at 0 and 24 hours showed measured concentrations ranged from less than the limit of quantification (LOQ, 0.001 mg/L) of the analytical method to 0.0790 mg/L. Analysis of the corresponding old media samples at 24 and 48 hours showed measured concentrations to range from <LOQ to 0.0173 mg/L.

Immobilisation observed during the second definitive test the effect was ≤10%. Statistical analysis showed that this immobilisation was not significant compared to the control.

Given that the test substance is acomplex UVCBsubstance and analytical monitoring was limited to thebranched dodecylbenzene aminefraction of the test substance, the effect concentrations were based on nominal loading rates.

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated under semi-static conditions and the 48-hour EL50 value was determined to be >1.0 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 1.0 mg/L, based on loading rate WAF.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated under semi-static conditions and the 48-hour EL50 value was determined to be >1.0 mg/L and the No Observed Effect Loading rate (NOEL) was determined to be 1.0 mg/L, based on loading rate WAF.