Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

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:

2017-10-16 to #

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:

adopted 13. Apr. 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

adopted 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal Concentration Test Item: Blank control, 0.1, 0.46, 1.0, 2.2, 4.6, 10 g/L resp. 0.10, 0.48, 1.07, 2.33, 4.83, 10.67 mL/L test item (based on a density of 0.94 g/mL stated in the MSDS).
- Sampling method: Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values).
- Sample storage conditions before analysis: not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test item is an UVCB substance, water-accommodated fractions (WAF) were prepared for the test by direct addition of the test material to the test medium. This was done by mixing the individual loading rates of 10 / 4.5 / 2.2 / 1 / 0.45/ 0.1 g/L, resp. 10.67 / 4.83 / 2.33 / 1.07 / 0.48 /. 0.10 mL/L test item (based on a density of 0.94 g/mL stated in the MSDS) with the corresponding amount of dilution water and stirring vigorously for 24 hours. After a settling period of 1 hour, the lower phase was used unfiltered as test solution. A settling bottle was used to receive the lower phase preventing the carry over of residues.
To avoid any loss of dissolved test item by membrane filtration, the solution was used unfiltered. The solution was not centrifuged because the test item is a liquid with a lower density than water.
- Differential loading: 10 / 4.5 / 2.2 / 1 / 0.45/ 0.1 g/L resp. 10.67 / 4.83 / 2.33 / 1.07 / 0.48 /. 0.10 mL/L test item (based on a density of 0.94 g/mL stated in the MSDS)
The loading rates were chosen upon results from non GLP pre-tests. In the pre-test the WAFs were prepared in the same way for loading rates of 0.1, 1 and 10 g/L. Because no toxicity was observed at the loading rate of 10 g/L in an unfiltered solution, this concentration was used as highest concentrated treatment in this study.

- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: bred in the LAUS GmbH throughout the year. Umweltbundesamt Berlin

husbandry is performed similar to the method described in the OECD guideline, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), Version 12 from 02. Feb. 2015.
- Feeding during test: no
- Type and amount of food: unicellular green algae (Desmodesmus subspicatus)


Other:
- Photo period: 16/8 hours, using neon tubes
- Medium: M4-Medium (recipe of ELENDT)
- Medium renewal: twice a week

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

19.5 – 21.5 °C

pH:

0 h: 7.5 - 7.7
48 h: 7.7 - 7.8

Dissolved oxygen:

0 h: 6.5 - 8.4
48 h: 8.2 - 8.3

Nominal and measured concentrations:

Nominal Concentration Test Item: Blank control, 0.1, 0.46, 1.0, 2.2, 4.6, 10 g/L resp. 0.10, 0.48, 1.07, 2.33, 4.83, 10.67 mL/L test item (based on a density of 0.94 g/mL stated in the MSDS).
Geometric Mean of measured concentrations: -, 27, 10, 10, 26, 40, 18 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers, glass, nominal volume 50 mL, tall shape.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 50 mL, tall shape
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4, each containing 20 ± 5 mL test solution and 5 daphnia
- No. of vessels per control (replicates): 4, each containing 20 ± 5 mL dilution water and 5 daphnia

TEST MEDIUM / WATER PARAMETERS
The newborn daphnia, aged between 0 and 22.5 hours, were sieved from the medium and immediately placed into a beaker containing dilution water. After a settling-in period of 85 minutes, animals which showed no apparent damage were used for the test. Switching from M4-medium (husbandry) to dilution water (test) has been shown to cause no detrimental effects for test daphnia.
- Source/preparation of dilution water: Dilution water (Daphnia medium) with the following specification will be used (stated in SOP 11800201):
- CaCl2*2H2O: 293.80 mg/L
- MgSO4*7H2O: 123.30 mg/L
- NaHCO3: 64.80 mg/L
- KCl: 5.80 mg/L
- Resulting hardness: 2.502 mmol/L
- Resulting hardness: 250 mg CaCO3/L
Deviations from the nominal weighted loads were less than 5 %. Exact values are recorded in the raw data. After preparation, the dilution water was aerated and the pH was measured. The pH was 7.7.

OTHER TEST CONDITIONS
- Adjustment of pH: not mentioned
- Photoperiod: 16 hours diffuse lighting / 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and mobility

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1 and 10 g/L
- Results used to determine the conditions for the definitive study: The loading rates were chosen upon results from non GLP pre-tests. In the pre-test the WAFs were prepared in the same way for loading rates of 0.1, 1 and 10 g/L. Because no toxicity was observed at the loading rate of 10 g/L in an unfiltered solution, this concentration was used as highest concentrated treatment in this study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7, CAS No. 7778-50-9)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 40 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate 4.6 g/L

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 40 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate 4.6 g/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 40 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate 4.6 g/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 40 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate 4.6 g/L

Details on results:

- Behavioural abnormalities: no
- Mortality of control: 1 daphnid was immobilised
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 24h-EC50: 1.6 mg/L
The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.

As the test item is an UVCB substance, water-accommodated fractions (WAF) were pre-pared for the test by direct addition of the test material to the test medium. This was done by mixing the individual loading rates of 10 / 4.6 / 2.2 / 1 / 0.46/ 0.1 g/L resp. 10.67 / 4.83 / 2.33 / 1.07 / 0.48 /. 0.10 mL/L test item (based on a density of 0.94 g/mL stated in the MSDS) with the corresponding amount of dilution water and stirring moderately for 24 hours. After a settling period of 1 hour the lower phase was used as test solution. A settling bottle was used to receive the lower phase preventing the carryover of residues (according to OECD guidance document no. 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, adopted 14. Dec. 2000; 3.11 [… Similarly any fraction of the mixture forming a surface film should be decanted to prevent surface trapping…]).

To avoid any loss of dissolved test item by membrane filtration, the solution was used unfiltered. The solution as not centrifuged because the test item is a liquid with a lower density than water. The test item did not show significant toxicity in the loading range tested. One of the animals was immobilised in the blank control and in the treatment with the highest loading rate of 10 g/L nominal.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using DOC-determination. The measured concentrations were strongly scattering (see chapter8.3) and correlation between nominal and measured concentration was weak because of the limited solubility of the test item and undissolved test material in some analytical samples. Due to the test items properties, a poorly soluble UVCB, the scattering measured DOC values are not unusual. The solubility of such a substance in test medium is not always comparable to the solubility in demineralised water in spite of the same handling and the same equipment as in a study for water solubility.

Table 1: Immobility.

Nominal concentration [g/L]	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
	0	0	0	0	0	0	0	0	1	5
Blank control	0	0	0	0	0	0	0	0	0	0
0.1	0	0	0	0	0	0	0	0	0	0
0.46	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0
2.2	0	0	0	0	0	0	0	0	0	0
4.6	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	1	0	0	0	5

Table 2: Measured Concentrations IC and TC.

Nominal Concentration Test Item	Measured TC t = 0 h	Measured TC t = 48 h	Measured IC t = 0 h	Measured IC t = 48 h
g/L	mg/L	mg/L	mg/L	mg/L
Blank control	10.49	19.76	9.68	11.45
0.1	33.66	30.37	9.46	10.75
0.46	18.58	18.54	9.43	10.55
1.0	19.73	18.28	9.83	10.55
2.2	34.94	27.96	9.49	10.79
4.6	43.85	40.27	9.52	10.68
10	29.38	21.18	9.33	10.43

LOQ (Limit of quantification) TC = 3.34 mg/L calibration from 12. Oct. 2017 (0 h)

LOQ (Limit of quantification) IC = 3.61 mg/L calibration from 12. Oct. 2017 (0 h)

LOQ (Limit of quantification) TC = 1.96 mg/L calibration from 23. Oct. 2017 (48 h)

LOQ (Limit of quantification) IC = 2.65 mg/L calibration from 23. Oct. 2017 (48 h)

Table 3: Measured Concentrations DOC

Nominal Concentration Test Item	Measured DOC (TC-IC) t = 0 h	Measured DOC (TC-IC) t = 48 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blanc control t = 48 h	Measured DOC t = 48 h
g/L	mg/L	mg/L	mg/L	mg/L	% of initial concentration
Blank control	0.81	0.81 *	--	--	--
0.1	24.20	19.62	23.39	18.81	80.4
0.46	9.15	8.00	8.34	7.19	86.2
1.0	9.91	7.73	9.10	6.92	76.0
2.2	25.45	17.17	24.64	16.36	66.3
4.6	34.33	29.58	33.52	28.77	85.8
10	20.05	10.75	19.24	9.94	51.7

* Because of a contaminated TOC-Vial, the measured value in the control after 48 hours was implausible high (8.31 mg/L). Therefore, the measured value in the control at the start of the test was used for the evaluation of the results.

Table 4: Calculated Test Item Concentrations and Geometric Mean.The geometric mean is calculated by multiplication of n participating concentrations and taking the n^throot.

Nominal Concentration Test Item	Calculated Concentration Test Item t = 0 h	Calculated Concentration Test Item t = 48 h	Geometric Mean of measured concentrations
g/L	mg/L	mg/L	mg/L
Blank control	--	--	--
0.1	29.94	24.07	27
0.46	10.68	9.20	10
1.0	11.65	8.86	10
2.2	31.54	20.94	26
4.6	42.90	36.82	40
10	24.63	12.72	18

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item against Daphnia magna was tested in a study conducted in accordance of the OECD guideline 202 and GLP. No significant toxic effects were observed. One of the animals was immobilised in the blank control and in the treatment with the 18 mg/L (geom. mean, meas).

Executive summary:

The 48–hr-acute toxicity of the test item to Daphnia magna Straus was studied under static conditions. Test species were exposed to control and test chemical at nominal concentration of 0.1, 0.46, 1.0, 2.2, 4.6, 10 g/L and geometric mean of measured concentrations: 27, 10, 10, 26, 40, 18 mg/L for 48 hr. Immobilisation and mortality were observed daily.  The 48–hour EC50 was > 40 mg/L (loading rate 4.6 g/L).  The 48–hr NOEC based on immobilization was ≥40 mg/L (loading rate 4.6 g/L).  One of the animals was immobilised in the blank control and in the treatment with 18 mg/L meas. No significant toxic effect was observed.

 As the test item is an UVCB substance, water-accommodated fractions (WAF) were pre-pared for the test by direct addition of the test material to the test medium. The WAFs were stirred moderately for 24 hours. After a settling period of 1 hour the lower phase was used as test solution. A settling bottle was used to receive the lower phase preventing the carryover of residues. Therefore, it is assumed that the test conditions represent the maximum solubility of the test item in the present test medium.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using DOC-determination. The measured concentrations were strongly scattering and correlation between nominal and measured concentration was weak because of the limited solubility of the test item. Due to the test items properties, a poorly soluble UVCB, the scattering measured DOC values are not unusual.

 

Based on the results of this study, VOELOFA Monomer would be classified as not toxic to Daphnia magna Straus in accordance with Section 4.1.2.6., Annex I of Regulation 1272/2008/EC.

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

 

Results Synopsis

 

Test Organism Age (e.g. 1^st instar): between 0 and 22.5 hours

Test Type (Flowthrough, Static, Static Renewal): Static

 

Geometric mean of the measured test item concentration:

48 -hr-NOEC: ≥ 40 mg/L (loading rate 4.6 g/L)                       

48 -hr-LOEC: > 40 mg/L (loading rate 4.6 g/L

24 -hr-EC₅₀ : > 40 mg/L (loading rate 4.6 g/L)

48 -hr-EC₅₀ : > 40 mg/L (loading rate 4.6 g/L)

Endpoint(s) Effected:  Immobility and mortality

Description of key information

The toxicity of the test item against Daphnia magna was tested in a study conducted in accordance of the OECD guideline 202 and GLP. No significant toxic effects were observed up to the water solubility (4.6 g/L loading corresponding to a meas. geom. mean of 40 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.6 g/L

Additional information

In a 48-h acute toxicity study, according to the OECD guideline 202, Daphnia magna were exposed to VOELOFA Monomer at nominal concentrations of 0.1, 0.46, 1.0, 2.2, 4.6, 10 g/L and geometric mean of measured concentrations of 27, 10, 10, 26, 40, 18 mg/L for 48 hr. The test solution was prepared by moderately stirring for 24 hours and it is assumed that the test conditions represent the maximum solubility of the test item in the present test medium. No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met. The result of the test is considered valid.