ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

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:

16 Oct 2017 - 06 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(EC) 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

EC Number:

500-276-7

EC Name:

ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

Cas Number:

89800-10-2

Molecular formula:

{(C10H16O7).(C3H3O)b.[(C6H10O2)m.(C3H3O)a]} (i.e., UVCB substance)

IUPAC Name:

ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and the 100 mg/L loading rate WAF test group from the bulk test preparation
- Sampling method: Samples were taken from the bulk test preparation at 0 h and from the pooled replicates at 72 h for quantitative analysis. Duplicate samples were taken at each occasion.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading:
200 mg test item was added to the surface of 2 L of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 h and the mixture allowed to stand for 1 h. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approx 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. An aliquot (1 L) of the WAF was inoculated with algal suspension (6.7 mL) to give the required test concentration of 100 mg/L loading rate WAF.
- Controls: Yes, only medium

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant agitation by orbital shaker (approx 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Culturing media and conditions (same as test or not): same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ±1 °C

pH:

7.6 - 9.3 (Control)
7.5 - 8.8 (100 mg/L)

Nominal and measured concentrations:

Control and 100 mg/L nominal loading rate (WAF)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (plugged with polyurethane foam bungs)
- Material, size, headspace, fill volume: 250 mL glass conical flasks were used; 100 mL of test preparation and 150 mL headspace
- Initial cells density: 0.5 x 10E+04
- Control end cells density: 163 x 10E+04
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: culture medium according the OECD 201 was prepared using reverse osmosis purified deionized water
- Intervals of water quality measurement: The pH of the control and 100 mg/L loading rate WAF was determined at initiation of the test and after 72 h exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: under continuous illumination
- Light intensity and quality: intensity approx 7000 lux, provided by warm white lighting (380 to 730 nm)
- Other: incubated (INFORS Multitron Version 2 incubator) and constantly shaken at approx 150 rpm for 72 h

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. Cell densities were determined after 0, 23, 50 and 72 h.
- Other: The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none, limittest
Range finding study
- Test concentrations: 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at 10 and 100 mg/L loading rate WAF.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): Whilst no abnormalities detected in the control cultures, some misshapen and shrunken cells were observed in the 100 mg/l loading rate WAF test cultures.

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
The ErC50 after 72 h was determined to be at 1.6 mg/L (95% confidence limits 1.4 to 1.8 mg/L) and the NOEC was observed to be at 0.25 mg/L.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate data after 72 h for the control and the 100 mg/L loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

Water Quality Criteria

Temperature was maintained at 24 ±1 ºC throughout the test. The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 9.3 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the EC Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Chemical Analysis of Test Loading Rates

Analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 2.59 mg/L was obtained. A decline in measured test concentration was observed at 72 hours to 1.03 mg/L indicating that the test item was unstable under the conditions of the test.Whilst a detectable level of test item was observed in the control culture at 0 hours (0.024 mg/L) this was considered to be due to post sampling contamination as this level was less than one hundredth of that found in the test preparation itself. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Table 1: Validity criteria for OECD 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	325	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	19%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1%	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The NOEL was 100 mg/L loading rate WAF.