Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug - 07 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 Jul 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg (10.12.2015).

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

EC Number:

614-257-7

Cas Number:

68071-40-9

Molecular formula:

C18H34O4, C20H38O4 and C22H42O4 (mainly)

IUPAC Name:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: One replicate per treatment without algae was run in parallel, which was used for the analytical sampling. Analytical samples were taken at 0 h (initial value) from fresh test solutions and after 72 h from aged solutions from all test loading rates and the control. For each sampling also a retain samples was taken.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared as water accommodated fractions (WAFs). A stock solution was prepared by directly weighing 10 mg into 1000 mL test medium. This stock solution was stirred in the dark at room temperature for 24 h (according to OECD guideline 23). The solution was clear and transparent but oily droplets floating on the surface of the solution were observed. Afterwards, the solution was filtered (cellulose nitrate membrane filter, 0.45 µm pore size), the transfer of oily droplets onto the filter was avoided. The test item loading rates were made by diluting the appropriate solutions with test medium without algae to give the required test loading rates. Since it was technically not possible to prepare the lower loading rates by direct weighing (amount too low), they had to be prepared by serial dilution (in accordance with OECD 23).
- Controls: 6 replicates without test item
- Evidence of undissolved material: No undissolved substance was observed but oily droplets floating on the surface of the stock solution were observed.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green microalgae
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Göttingen, Germany
- Age of inoculum (at test initiation): 3 - 4 d
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solutions in order to keep the algae in an exponential growth state. Culture conditions: Illumination: 60 - 120 µEm^-2s^-1; Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks. CO2 was supplied through the shaking of the flasks on a rotating shaker (105 rpm).
Stock cultures are ordered regularly from the supplier. For the test, a pre-culture in the state of exponential growth was prepared 3 - 4 d before start of the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.3 - 23.0 °C

pH:

0 h: 7.28 (control), 7.27 - 7.31 (treatments)
72 h: 7.74 (control), 7.68 - 8.05 (treatments)

Nominal and measured concentrations:

Control, 0.0954, 0.305, 0.977, 3.13, and 10 mg/L (nominal loading rate)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks filled with 50 mL test medium.
- Type : Closed with aluminium caps.
- Initial cells density: 0.5 E4 cells/mL (nominal), 0.64 E4 cells/mL (average actual concentration)
- Control end cells density: 42.63 E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, AAP-medium according to Annex 3, OECD 201. The pH of the test medium was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 73.2 - 83.8 µE*m^-2*s^-1 at cell culture level. The mean light intensity for all positions was 79.7 µE*m^-2*s^-1 at test start with measured values deviating in a range of + 5.1 to -8.2%, which was within ± 15% of variation, as specified by OECD 201.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell numbers were derived from the fluorscence signals, which were measured by fluorimeter (infinite 200Pro), with an emission wavelength of 670 nm, equipped with Tecan i-control software for Tecan Readers (i-control, 1.11.1.0)
- Other: The morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

8.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 7.61 - 9.49 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

After 72 h no clear concentration response relationship was observed for loading rates 0.0954 – 3.13 mg/L. At the nominal loading rate of 3.13 mg/L no inhibition was observed for growth rate or yield. At the nominal loading rate of 10.0 mg/L the inhibition of growth rate peaked at 64.2% and the inhibition of yield peaked at 93.5% (Table 2 in "Any other information on results incl. tables").

- Exponential growth in the control: Yes, cell numbers in the control increased by a factor of 66.61 after 72 h, which corresponds to a growth rate of 1.4 d^-1.
- Observation of abnormalities: Some spherical cells were observed at 10.0 mg/L nominal test item loading rate.
- Effects above the solubility of substance in test medium: Yes, it is possible that physical effects of the test item caused the observed inhibition based on substance characteristics. The test item has an amphiphilic structure and surface-active properties leading to an increased potential to cause physical effects in aquatic studies.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. The routine toxic reference test was performed from 20 - 23 Jun 2016 and fulfilled the validity criteria.
- ErC50 (72 h): 1.16 mg/L (nominal)

Reported statistics and error estimates:

The statistical evaluation for the 72 h period was performed for growth rate and yield using SAS (2002 - 2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for yield, Bonferroni-Holms corrected Welch test, left sided, for growth rate). The EL50 values for growth rate and yield were calculated using Spearmann-Kaerber estimator (Anellis & Werkowsky, 1968). The EL10 and EL20 values could not be determined from the obtained data. The NOELR was calculated by the Bonferroni-Holms corrected Welch test for growth rate and Dunnett-t-test for yield (left sided, p < 0.05). For statistical reasons inhibition values below 0% were set to zero.

Any other information on results incl. tables

ANALYTICAL RESULTS

The test item is a UVCB consisting of several components with similar chemistry. No adequate analytical method for a sufficiently accurate quantitative analysis of the individual components was available. As indication of the concentrations of test item in the respective test solutions, the content of total organic carbon (TOC) in the solutions was measured. In addition, the TOC content of the test item as a bulk material was determined.

TOC analysis proved the presence of the test item in the solutions but was found to be unsuitable for quantitative purposes. Thus, the effects were evaluated based on the nominal loading rates. The measured initial concentration ranged from < LOQ to 14.1% of nominal loading rate. In the aged samples the measured content was between < LOQ and 10.9% of nominal loading rate (Table 1). The low recoveries are consistent with the low water solubility of the test item (1.9 mg/L at pH 6.13 and 19.6 – 20.0 °C).

  

Table 1. TOC measurement.

Loading rate nominal [mg/L]	TOC content^1 nominal [mg/L]	Sampling	TOC measured in test solution^2 [mg/L]	TOC content % of nominal
Control	0	0 h fresh	< LOQ	-
		72 h aged	< LOQ	-
0.0954	0.0651	0 h fresh	< LOQ	-
		72 h aged	< LOQ	-
0.305	0.208	0 h fresh	< LOQ	-
		72 h aged	< LOQ	-
0.977	0.666	0 h fresh	< LOQ	-
		72 h aged	< LOQ	-
3.13	2.13	0 h fresh	< LOQ	-
		72 h aged	< LOQ	-
10.0	6.82	0 h fresh	0.960	14.1
		72 h aged	0.740	10.9

- = not calculated; LOQ = 0.300 mg/L TOC

¹= Based on analysed TOC-content of test item (mass of carbon was 68.2% of total mass)

²= control corrected

 

BIOLOGICAL RESULTS

            

Table 2. Percentage inhibition of growth rate.

Loading rate [mg/L]	% inhibition of growth rate
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.0954	-4.5	-0.6	0.4^1)
0.305	1.2	0.6	1.3^1)
0.977	2.5	2.0	2.7^1)
3.13	-1.2	-6.0	-4.1^2)
10.0	94.7	92.0	64.2 *

¹⁾Value was omitted for EC50 calculation.

²⁾Value was set to zero for EC50 calculation.

* Statistically significant difference to the control.