Registration Dossier

Ecotoxicological information

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
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test Item: Baccartol Crude
Batch Number: VE00364716
Purity: Not applicable - complex mixture
Appearance: yellow viscous liquid
Expiry date: 2017-04-02
Recommended storage: Keep container tightly closed in a dry and well-ventilated place at ambient temperature (10 - 30 °C) protected from light.

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Details on test solutions:
Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important technical groups in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the equilibrated aqueous phase, i.e. the WAF. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAF) were prepared with loadings of the test item in the range of 3.125 to 50.0 mg/L: 3.125 - 6.25 - 12.5 - 25.0 - 50.0 mg/L. The loading levels are based on the non-GLP preliminary range finding test.
For each loading level, an appropriate amount of the test item was pipetted into a brown glass flask with an appropriate amount of dilution water (see Table 2). These dispersions were shaken for 24 hours at 20 rpm at room temperature. After a separation phase of 1 hour, the WAF’s were removed by siphoning (from the approximate bottom of the flask) and used in the test. The WAF’s were checked via laser beam (Tyndall effect) for undissolved particles.

Test loading
Per definition of the WAF all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
Reason for the selection of the test organism: Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.
Origin: Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567-5130 lux for 24 hours per day.
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h

Test conditions

Hardness:
The medium had a nominal hardness of 0.24 mmol Ca+Mg/L.
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2 °C.
pH:
The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control after measurement of the cell density.
Nominal and measured concentrations:
Range finding Test
Nominal: 1, 10 and 100 mg/l
Measured at 0 h: 0.850, 11.7 and 98.7 mg/l
Measured at 72 h: 0.830, 2.47 and 75.9 mg/l

Definitive Test
Nominal: 3.125, 6.25, 12.5, 25.0, 50.0 mg/L
Geometric mean measured: 1.88, 5.28, 7.64, 18.0 nad 39.1 mg/l

The measured concentrations of BACCARTOL CRUDE in the fresh media (0 h) were in the range of 88 to 113% of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 37 to 63 % of the nominal values of all concentrations.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
9.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
5.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Effects based on yield were also reported (see attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table. Furthermore, the preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus, only the EL10 is presented in the above "effects concentration" table. The NOEC values are available in the full study report.
Results with reference substance (positive control):
The toxicity of potassium dichromate (Sigma, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace) and 2016- 10-10 to 2016-10-13 (without headspace), respectively. The reference item toxicity is in the valid range following test facility SOPs.

Closed test vessels without headspace were used to mimic testing a volatile test item and results were compared with the standard system in open vessels. The data of both set-ups are consistent. It is evident from these results that the use of a modified test system to reduce losses of the test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system. As such it can be considered that the results from the definitive test give a reliable result for the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, BACCARTOL CRUDE was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal loading levels):
The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 9.12 (6.25 - 12.5) mg/L and 6.54 (3.125 - 12.5) mg/L, respectively. The EL10-values for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 5.67 (3.125 - 6.25) mg/L and <3.125 mg/L, respectively. The NOEL-values for inhibition of growth rate and yield after 72 hours were both < 3.125 mg/L.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EL50 and EL10 based on growth rate are used for classification purposes, which were determined in this study to be 9.12 mg/L and 5.67 mg/L respectively.