Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.04.2019 - 22.07.2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Raphidocelis subcapitata Growth Inhibition Test according to OECD 201 (23 March 2006)

Deviations:

no

GLP compliance:

yes

Remarks:

Substance specific analysis was performed without GLP.

Specific details on test material used for the study:

- Source and package No.of test material: lot #19953859
- Expiration date of the lot/batch: 16.01.2020

Analytical monitoring:

yes

Details on sampling:

Nominal test item loading rate [mg/L]: 0 - 0.5 - 1.0 - 2.0 - 4.0 - 8.0
Sampling method:
After 0 h and 72 h exposure, the additional vessels of NC, C, D and E were sampled: 4 samples of 7 mL per treatment group and time. The samples were filled into 15 mL centrifuge tubes and stored in the refrigerator (2 - 8°C). Item concentrations were verified via LC-MS and TOC.

Vehicle:

yes

Remarks:

OECD TG 201

Details on test solutions:

Solution A1
Ammonium chloride p.a. NH4Cl 1.5 g
Magnesium chloride p.a. MgCl2 × 6H2O 1.2 g
Calcium chloride dihydrate p.a. CaCl2 × 2 H2O 1.8 g
Magnesium sulfate heptahydrate p.a. MgSO4 × 7H2O 1.5 g
Potassium dihydrogen phosphate p.a. KH2PO4 0.16 g
dissolved in 1000 mL ultrapure water
Solution A2
Iron (III) chloride hexahydrate p.a. Fe(III)Cl3 × 6H2O 0.0640 g
Sodium-EDTA dihydrate Na2 EDTA × 2H2O 0.1000 g
dissolved in 1000 mL ultrapure water
Solution A3
Boric acid p.a. H3BO3 0.185 g
Manganese chloride tetrahydrate p.a. Mn(Cl 2) × 4H2O 0.415 g
Sodium molybdate dihydrate p.a. Na 2 MoO4 × 2H2O 0.007 g
Zinc chloride p.a. ZnCl2 0.003 g
Cobalt chloride hexahydrate p.a. CoCl2 × 6H2O 0.0015 g
Copper chloride dihydrate p.a. CuCl2 × 2H2O 0.00001 g
dissolved in 1000 mL ultrapure water (for ZnCl2 , CoCl2 and CuCl2 higher concentrated solutions were prepared and the correspondent volumes used for A3).
Solution A4
Sodium hydrogen carbonate p.a. NaHCO3 50 g
dissolved in 1000 mL ultrapure water. Solutions A1 – A3 were autoclaved, solution A4 was sterile-filtered (pore diameter 0.22 µm).

A 10-fold concentrated medium was prepared by using 100 mL of stock solution A1 and 10 mL of stock solutions A2, A3 and A4, filling up to 1000 mL with ultrapure water and aerating for 1.5 h. The 1-fold concentrated medium was prepared by diluting 300 mL of the 10-fold concentrated medium with 2700 mL ultrapure water.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism Raphidocelis subcapitata (Strain No.61.81SAG) originates from the Culture Collection of Algae at the University of Goettingen. The strain used for this study has been cultured in suspension culture at Hydrotox GmbH since May 2018. Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium (composition: 496 mg/L NaNO3 , 39 mg/L K2HPO4, 75 mg/L MgSO4 ×7H2O, 36 mg/L CaCl2 ×2H2O, 58 mg/L Na2CO3 , 10 mg/L Na 2 EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2 ×4H2O, 0.0088 mg/L ZnSO 4 ×7H2O, 0.0032 mg/L CuSO4 ×5H2O, 0.0010 mg/L Na2MoO4 ×2H2O, 0.0016 mg/L CoCl2 ×6H2O) under axenic conditions to keep it in exponential growth.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

22.1 - 23.3 °C

pH:

7.7

Nominal and measured concentrations:

Nominal: 0.5, 1.0, 2.0, 4.0, 8.0
Measured: 0.0501, 0.3581, 0.4189, 0.5672

Details on test conditions:

Light intensity: 84.4 µE m - ² s -1

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

other: ELR50

Remarks:

Growth rate

Effect conc.:

>= 4.184 - <= 4.184 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Details on results:

The test is valid according to OECD Test Guideline 201 (23 March 2006, corrected 28 July 2011).
The Algae Growth Inhibition Test was performed as water accommodated fraction (WAF) with five Loading Rates (LR) between 0.5 mg/L and 8 mg/L. The results are given in relation to the nominal test item Loading Rate.
TOC analysis : The measured mean TOC concentrations in the test item treatments were below the limit of quantification (LOQ: 1.619 mg/L; NPOC-method).
Substance specific analysis without GLP :
As reaction components of the test item (UVCB) which are presumably responsible for the effects on algae, 2 key components (N-(3(dimethylamino)propyl)palmitamide and N-(3(dimethyl-amino)propyl)stearamide) could be detected in the test item treatments, but the measured values were below the limit of quantification (LOQ: 5 µg/L).
Cell morphology: The morphology of the algal cells in the control and in the test item concentrations showed no obvious abnormality. Due to the toxic effect in the highest concentrations of D and E some dead algae cells were visible.
The effect concentrations after 72 h exposure were calculated with the statistical software ToxRat Professional 3.2.1 (Table 3; Appendix V). All results are given in relation to the nominal test item loading rates. With the loading rates between 0.5 to 2.0 mg/L, the test item showed rather a growth promoting effect than a growth inhibiting effect. With the loading rates of 4.0 and 8 mg/L a steep increase of growth inhibiting effects could be observed (see sections 6.2.3 & 6.2.4). Due to the steep dose-effect relationship no confidence limits could be derived.
Test parameters:
• The pH was 7.7 in the control and 7.7 – 7.8 in the test item treatments (Table 4; required:increase ≤ 1.5).
• The temperature during the exposure was 22.1 – 23.3 °C (required: 21 - 24 °C, constant within ± 2 °C).
• The lighting was continuous with an intensity of 84.4 µE m - ² s -1 ± 8.7 %. (required: 60 – 120 µE m - ² s -1 ).

Results with reference substance (positive control):

ErC50 (72h) 1.15 mg/L (CL 95%: 1.15 - 1.34 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The 72h ELR20 and ELR50 are 3.963 and 4.184 mg/L based on the loading rate. The substance is not classified under CLP regulation.

Executive summary:

In the Klimisch 1 GLP study the toxicity of test item to Raphidocelis  subcapitata as determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201.

The Algae Growth Inhibition Test was performed as water accommodated fraction (WAF) with five Loading Rates (LR) between 0.5 mg/L and 8 mg/L. The results are given in relation to the nominal test item Loading Rate. The 72h ELR20 and ELR50 are 3.963 and 4.184 mg/L based on the loading rate. The test is valid according to OECD Test Guideline 201 (23 March 2006, corrected 28 July 2011). The substance is not classified under CLP regulation.