Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized

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:

28-Oct-2016 to 02-Dec-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

Principles of method if other than guideline:

Based on Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000, as the substance is an UVCB the test item preperations for testing were prepared using the water accomated fraction (WAF) approach.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Confidential
- Expiration date of the lot/batch: 30-Dec-2017
- Purity test date: 100 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature
- Stability under test conditions: Sufficient for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: None
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved for 3 days to make a water accomated fraction in test medium
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L (100 mg/L measures were conducted with and without algae).
- Sampling method: For sampling 4.0 mL from the approximate centre of the test vessels were taken at 0, 24 and 72 hours. At the end of each exposure period the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15°C) until analysis; stability at this storge temperature has been previously confirmed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accomadated fractions at 1.0, 10 and 100 mg/L were prepared seperately in medium with no solvent. The WAFs were prepared by adding known amounts of test item to the medium and allowing it to equilibrate over 3 days, stirring continuously. Following cessation of stirring and one hour of settling the aqueous phase was siphoned off for testing.
- Eluate: N/A
- Differential loading: N/A
- Controls: Test medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None, dissolved material was checked for using Tyndall's effect and no undissolved material was found.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL-1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1 medium) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s) in a climate controlled room at a temperature of 21-24 °C.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Testing conditions are the similar, however M2 medium is used in favour of M1 medium.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with guideline.

Post exposure observation period:

N/A

Hardness:

24 mg CaCO3/L

Test temperature:

22 to 23 °C

pH:

8.1 - 8.3

Dissolved oxygen:

Not reported

Salinity:

N/A

Conductivity:

Not reported

Nominal and measured concentrations:

Concentrations were based as a loading rate and were 1.0, 10 and 100 mg/L. The measured concentraion at 100 mg/L was 3.5 mg/L at 0 hours and remained => 64 % of initial concncetrations for the duration of the test. The endpoints were represented as loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all glass
- Type (delete if not applicable): capped
- Material, size, headspace, fill volume: Vessels were filled with 50 mL of test solutions
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate):N/A
- Initial cells density: 10^4
- Control end cells density: Mean = 151.9 x 10^4 cells (St. Dev. 27.7 x 10^4 cells)
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 6 for the 100 mg/L group, 3 for the 1.0 and 10 mg/L groups
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: M2-medium was used.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

- Culture medium different from test medium: Yes, M1 Culture medium:
according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: Not reported
- Conductivity: N/A
- Culture medium different from test medium: N/A
- Intervals of water quality measurement: Intervals of water quality measurement: At the beginning and end of the test pH was measured. Temperature was monitored continuously in the control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 84 to 89 μE.m-2.s^-1.
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning of the test cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometer with an immersion probe (at 680 nm, pathlength = 20 mm). Algal medium was used as a blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- Chlorophyll measurement: N/A
- Other: At the end of the test microscopic observations were performed on the control and the WAF at 100 mg/L to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.0, 10 and 100 mg/L (Combined limit/range-finder test)
- Justification for using less concentrations than requested by guideline: Combined limit/range-finder test
- Range finding study: Combined limit/range-finder test
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No definitive study required due to lack of effect.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Unusual cell shape: none observed
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: N/A
- Any stimulation of growth found in any treatment: minimal and non-biologically relevant stimulation in growth and yield was found in WAFs prepared at 1.0 and 10 mg/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: uknown for growth rate.

Results with reference substance (positive control):

- Results with reference substance valid?
- 72h ErC50: > 100 mg/L
- 72h NOErC: => 100 mg/L
- 72h EyC50: > 100 mg/L
- 72h NOEyC: 100 mg/L
- Other: N/A.

Reported statistics and error estimates:

The 72h-ErC50 and EyC50 was > 100 mg/L. No statistical analysis is needed as no/minimal effects were seen.

An effect was considered to be significant if statistical analysis of the data obtained for the loading rates compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one- sided). The calculations were performed with ToxRat Professional v. 3.2 (ToxRat Solutions® GmbH, Germany).

Yield was significantly reduced at a WAF of 100 mg/L as compared to the control. The reduction was seen at 36.9 %.

Percentage inhibition of growth rate (total test period)

Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.670	0.0637	6
1.0	1.696	0.0199	3	-1.6
10	1.677	0.0168	3	-0.5
100	1.522	0.0198	6	8.8^#

^#Effect was statistically significant but not biologically relevant (<10%)

Percentage inhibition of growth rate at different time intervals

Loading rate (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.646	0.0	1.799	0.0	1.564	0.0
100	6	1.520	7.7	1.591	11.5	1.455	7.0

     
Percentage inhibition of yield

Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	150.9	27.68	6
1.0	161.5	9.64	3	-7.0
10	152.4	7.83	3	-1.0
100	95.3	5.64	6	36.9*

* Effect was statistically significant

pH levels recorded during the final test

Loading rate (mg/L)	pH
	t=0h	t=72h
Control	8.2	8.3
100	8.1	8.2

Validity criteria fulfilled:

yes

Remarks:

Validity criteria was met as outlined by the used guidelines.

Conclusions:

Under the conditions of the study with Pseudokirchneriella subcapitata, the test item inhibited growth rate and yield of this fresh water algae species by 9 and 37%, respectively, when the algae were exposed to a water accommodated fraction prepared at a loading rate of 100 mg/L.

The EL50 for inhibition of both growth rate (72h-ERL50) and yield (72h-EYL50) was >100 mg/L.
The 72h-NOELR for growth rate inhibition was 100 mg/L, while the 72h-NOELR for yield inhibition was <100 mg/L.

Executive summary:

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 2016/266, 2015 and the OECD series on testing and assessment number 23, 2000.

 

The test item was an off white paste and a UVCB. The test item was not completely soluble in test medium at the loading rates initially prepared.

 

All test concentrations were prepared separately as water accommodated fractions (WAFs) due to the UVCB nature of the test item. Measured amounts of the test item were added directly to test medium and mixed for three days to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in the aqueous phase. Following cessation of mixing and a one hour period of settling (to allow phase separation) the aqueous phase, i.e. the WAF, was siphoned off for testing. Just before testing the WAFs were checked for tyndall effect, using a Laser pen.

 

A combined limit/range-finding test was performed exposing six replicates of exponentially growing algal cultures to a control and a WAF at 100 mg/L. In addition, three replicates per group were exposed to WAFs at 1.0 and 10 mg/L. The initial algal cell density was 104 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

 

Analysis of the samples taken from the WAF at 100 mg/L showed a measured concentration of 3.5 mg/L at the start of the test. During the exposure period the measured concentration decreased to 64% of initial at the end of the 72-hour test period. However, analysis was based on thesum of the peak area of the two main large peaks in the chromatograms and are therefore only representative of the actual concentration of the entire UVCB. Therefore,the effect parameters were based on the initial loading rate instead of measured concentrations.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The test item inhibited growth rate and yield of this fresh water algae species by 9 and 37%, respectively, when the algae were exposed to a water accommodated fraction prepared at a loading rate of 100 mg/L.

 

The EL50for inhibition of both growth rate (72h-ERL50) and yield (72h-EYL50) was >100 mg/L based on nominal loading rates to the test system as water accommodated fractions.

 

The 72h-NOELR for growth rate inhibition was 100 mg/L, while the 72h-NOELR for yield inhibition was <100 mg/L based on nominal loading rates to the test system as water accommodated fractions.

Description of key information

72 h ErL50 (Pseudokirchneriella subcapitata) > 100 mg/L, 72 h NOErLR (Pseudokirchneriella subcapitata) => 100 mg/L, OECD 201 Freshwater alga and cyanobacteria, growth inhibition test (Anon., 2017).

Additional information

The EL50for inhibition of both growth rate (72h-ERL50) and yield (72h-EYL50) was >100 mg/L based on nominal loading rates to the test system as water accommodated fractions.

 

The 72h-NOELR for growth rate inhibition was 100 mg/L, while the 72h-NOELR for yield inhibition was <100 mg/L based on nominal loading rates to the test system as water accommodated fractions.