A mixture mainly based on: 2,3-dihydro-6-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene; 2,3-dihydro-5-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

not specified

GLP compliance:

yes

Vehicle:

no

Details on test solutions:

Water accomodation fraction approach

Test organisms (species):

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

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Basis for effect:

biomass

Details on results:

% Concentration loss over test: ... 85
The test item was light sensitive and its concentration was not maintained over 80% of the initial value throughout the duration of treatment. A clear NOEC cannot be defined, nevertheless the data indicated the exposure to the test item and its degradation products resulting from photolysis, caused no adverse effects in the algae under the reported experimental conditions.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 2014 - 23 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage: Room temperature

Analytical monitoring:

yes

Details on sampling:

Sampled at 0 and 72 hours
Samples, properly diluted with deionised water with low TOC contents, have been analysed by means of an integrated sampling system directly from vials with 40 mL volume.

Vehicle:

no

Details on test solutions:

The test sample has been prepared at the maximum solubility because the test item is a solid, very poorly soluble in water. The test item has been melted (at 70°C for 1/2 hour) then about 200 mg have been put in culture medium (1000 ml) and to facilitate its solubility in water (components soluble in water) the stock solution has been stirred (10 rpm) for 72 hours and then before the beginning of the test this solution has been filtered (0.45 pm). Then 5 different concentrations in water has been tested.

The total volume of the culture medium and the concentration of the algae lnoculum added are thesame in all vessels.

Test organisms (species):

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

Details on test organisms:

Test organism: Green alga
Family: Clorophyceae
Order: Chlorococcalles

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period

Hardness:

0.6 g CaCl2 2H20 in 500 mL water

Test temperature:

23 ± 2 °C

pH:

7.5 - 9.0

Dissolved oxygen:

Not measured

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal : 200, 133.33,. 88.89, 59.26, 39.51 mg/L; actual concentrations not measured

Details on test conditions:

TREATMENT
Algal suspension, coming from stock culture, has been placed in the receivers containing the defined concentration of the test item.
Erlenmeyer flasks containing treated organisms and Erlenmeyer flasks containing control organisms, have been kept in suspension by means of steady stirring at a temperature of 23°C i2°C with continuous lightning for a total period of 72 hours.

SAMPLE PREPARATION
The test sample has been prepared at the maximum solubility because the test item is a solid, very poorly soluble in water. The test item has been melted (at 70°C for 1/2 hour) then about 200 mg have been put in culture medium (1000 ml) and to facilitate its solubility in water (components soluble in water) the stock solution has been stirred (10 rpm) for 72 hours and then before the beginning of the test this solution has been filtered (0.45 pm). Then 5 different concentrations in water has been tested.
The total volume of the culture medium and the concentration of the algae lnoculum added are the same in all vessels.

OBSERVATIONS
Every 24 hours from the beginning of the test until the end of the test (72 hours), cellular concentration has been determined, by means of Burker chamber, of treated and of control organisms. pH has been measured at the beginning and at the end of the assay.

CULTURING OF ALGAE
The algal suspension has been kept in culture medium in sterile 250 ml Erlenmeyer flasks with the
following conditions:
Temperature: 23°C ± 2°C
Lighting: lighting greater than 4300 lux
Stirring: Steady with electrical stirrer

ASSAY CONDITIONS
Starting cellular density: 10E+04
No of replications: 3 (6 for the control)
Concentrations: 5
Lightning: Continuous — with fluorescent lamp 8000 lux
Stirring: Steady with electrical stirrer
Temperature: 23°C ± 2°C
Assay duration: 72 hours

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

112 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

A normal and healthy appearance of the algae at the end of the test was observed at microscopic

Results with reference substance (positive control):

No reference standard (positive control)

EQUATIONS

For each sample, the corresponding blank is analysed. The result is expressed corrected from the blank inoculum and the dilution water at initial and final timing test.

TOC substance (t=0) = TOC substance _t=0 — TOC (blank _t=0 - dilution water _t=0)

TOC substance (t) = TOC substance _t — TOC (blank _t— dilution water _t)

% stability = (TOC substance t/ TOC substance t=0 ) x 100

The sample is considered stable if the % stability is between 80—120 % of the value.

Average growth rate:

The average growth rate for a specific period (µ _i-j) is calculated as the logarithmic increase in the number of cells from the following equation for each control and treated vessel:

Calculate the average specific growth rate over the entire period using the nominally inoculated number of cell/mL as the starting value (X_i)

µ_i-j = (ln X _j - ln X _i) / (t_j - t_i)

Where

µ_i-j = is the average specific growth rate from time i to timej

X_i = the number of cell/mL at time i

X_j = the number of cell/mL at time j

The percent inhibition of growth rate was calculated as follows:

%I_r = ((µc - µt) / µc) x 100

VALIDITY CRITERIA

The assay is valid if the following criteria are satisfied:

- the cell concentration in the control cultures should have increased by a factor of at least 16 within three days corresponds to a specific growth rate of 0.92 day ^-1.

- the mean coefficient of variation for section by section (CV_ss) specific growth rates in the control cultures must not exceed 35%

- the coefficient of variation of average specific growth rates during the whole period (CV_wp) in replicate control cultures must not exceed 7%.

- pH must not vary for more than a 1.5 units during the assay

Validity criteria fulfilled:

yes

Conclusions:

The obtained results, in compliance with assay validity criteria, showed that Algae EC50 after 72 hours of the nominal concentration of the test item is 112 mg/l.

Analytical verification of the concentration of the test item - The test item shouldn’t be considered stable, because the TOC values, at the beginning and at the end of the test didn’t remain within the required range (SO-120%) at the higher tested concentration. The percentages of stability are reported in the Addendum N.1- Table N.4.

Validity criteria:

Cellular concentration - Algae densities for treated and control during the assay are reported in Addendum N. 1—Table N.1 A normal and healthy appearance of the algae at the end of the test was observed at microscopic

pH - The results of pH at the beginning and at the end of the test are reported in Addendum N. 1— Table N2 and satisfy the requirements of the regulation (pH must not deviate by more than 1.5 units during the test).

Temperature - The results of temperature during the whole test are reported in Addendum N. 1-Table N. 3 and satisfy the requirements of the guideline (23 ± 2°C).

Executive summary:

On the extract of the test item the growth inhibition test on a culture of algae Pseudokirchneriella subcapitata was performed on a culture in exponential growth phase.

This approach, according to OECD 201, allowed to calculate the growth inhibition (effect) concentration to the 50 % of the organisms (EC50).

The algae have been exposed to 5 different dilutions of the test item for a total period of 72 hours. After 72 hours of exposition, cellular concentration has been measured using a Burker chamber.

At the end of the test, microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.

At the beginning and at the end of the test (72 hours), pH and temperature have been measured in all the vessel. Organic carbon concentration (TOC) have been measured at the beginning and at the end of the test in a pool derived from vessels at the higher, intermediate and lower tested concentration.

Summary: EC50 (72h) - 112 mg/L.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

112 mg/L

EC10 or NOEC for freshwater algae:

53 mg/L

Additional information

The substance does not meet the criteria for classification in accordance with the classification, labelling and packaging regulation (EC 1272/2008).