Reaction mass of 3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-4-yl)propanal

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:

25th to 31st January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 2013-06-03/05. Date of signature: 2013-11-05

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal: 0 (control), 1.6, 3.1, 6.2, 12.5, 25.0 and 50.0 mg/l.
- Sampling method: Separate replicates for the test item analysis at the beginning of the exposure were prepared with algae. For the test item analysis after 24, 48 and 72 hours samples were taken out of the test replicates. If volumes of the test medium were insufficient, separate replicates
were prepared with algae at the beginning of the exposure and incubated under test conditions.
- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For each test concentration, an appropriate amount of the test item was weighed and transferred in graduated flask with an appropriate amount of dilution water. The test concentrations were pre-incubated at 30 ± 2 QC with constant stirring with approximately 1000 rpm for 24 hours. Thereafter the stock solution was allowed to stand at 21 - 24 QC (test temperature) for 16 - 24 hours.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: HINDAK, SAG 61.81
- Source (laboratory, culture collection): Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universitat Gottingen, Nikolausberger Weg 18, D-37073 Gottingen. Cultivation at test facility.
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks prepared every month on Z-Agar.

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LOTTGE et al. (1994).
- Any deformed or abnormal cells observed: Not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 24 QC, controlled at ± 2 QC

pH:

7.34 - 8.76

Dissolved oxygen:

6.95 - 8.73

Nominal and measured concentrations:

Nominal: 0 (control), 1.6, 3.1, 6.2, 12.5, 25.0 and 50.0 mg/l.
Measured: 0 (control), 0.24, 0.326, 1.09, 6.54, 16.35 and 36.67 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Type: closed: sealed with cotton wool plugs
- Material, size, headspace, fill volume: 250 ml flask, no headspace
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Static
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL
Current: 7085 cells/mL
- Control end cells density: 1941419 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Threefold concentrated AAP medium according to the guidelines).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Component Concentration [mg/L]
NaN03 76.5
MgCI2·6 H2O 36.492
CaC12·2 H2O 13.23
MgS04' 7 H2O 44.1
K2HP04 3.132
NaHC03 45
H3B03 0.5565
MnCI2· 4 H2O 1.246
ZnCI2 9.81 x 10-3
COCI2·6 H2O 4.29 x 10-3
CUCI2·2 H2O 0.036 x 10-3
NaMo04' 2 H2O 21.78 x 10-3
FeC13·6 H2O 0.4794
Na2-EDTA 0.9
pH 7.5±0.1

- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value and the dissolved oxygen concentration was measured daily. At the start of the exposure and after 24 and 48 hours pH and dissolved oxygen were measured in one additional replicate of each test item concentration and the control. At the end of the exposure, they were measured in a pooled sample of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: Yes
- Adjustment of pH: The pH of the test medium has to be 7.5 ± 0.1 and was adjusted prior to testing with addition of 1 M NaOH or HCl.
- Photoperiod: 24 hour day/light
- Light intensity and quality: 60 - 120 uE . m-2 . S-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density daily.

- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH) for determination of the cell density.

- Chlorophyll measurement:Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the preliminary test at the nominal concentration level of 100 mg/l.

- Other: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

- Range finding study
- Test concentrations: 1, 10, 100 mg/l
- Results used to determine the conditions for the definitive study: 4 % Growth rate inhibition at 1.0 mg/l and 100% inhibition at 100 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dicromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL 3.60- 12.8 mg/l

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 2.98 - 6.52 mg/l

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.326 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 1.07 - 1.52 mg/l

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.421 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 0.351 - 0.526 mg/l

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.
- Unusual cell shape: None reported
- Colour differences: None reported
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Any stimulation of growth found in any treatment: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.749 mg/l growth rate (valid range: 0.821 ± 0.388 mg/l)
- Other: EyC50: 0.282 mg/l (valid range: 0.429 ± 0.261 mg/l)

Reported statistics and error estimates:

The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures. Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.

Table 1: Growth Rate Inhibition using Nominal Concentrations

Nominal concentration [mg/L]	Growth Rate Inhibition [%]
50.0	100
25.0	100
12.5	42
6.25	10
3.13	2
1.62	1

Table 2: Geometric mean measured concentrations where 0.05 mg/l is used in the calculation in place of <LOQ

Nominal concentration [mg/l]	1.60	3.10	6.20	12.5	25.0	50.0
After 0 h	1.57	2.88	6.81	13.6	25.1	48.0
After 24 h	0.856	1.57	4.87	11.9	20.7	42.1
After 48 h	0.05	0.05	0.852	8.78	16.3	42.2
After 72 h	0.05	0.05	0.05	1.29	8.44	21.2
Geomean concentration [mg/l]	0.24	0.33	1.09	6.54	16.35	36.67

Validity criteria fulfilled:

yes

Conclusions:

A 72h-EC50 value of 6.75 mg/L, a 72h-EC10 value of 5.41 mg/L and a 72h-NOEC value of 0.326 mg/L were determined based on growth rate and geometric mean measured concentrations, in a reliable study of toxicity to Pseudokirchneriella subcapitata, conducted according to an appropriate test protocol, and in compliance with GLP.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the EU Method C.3 and the OECD Guideline 201 with GLP statement, under static conditions over a period of 72 hours, with an initial cell density of 7085 cells/ml. Six concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 1.60- 3.10- 6.20 - 12.5 - 25.0 - 50.0 mg/L corresponding to geometric mean measured test item concentrations of 0.241 - 0.326- 1.09- 6.54- 16.4- 36.7 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The concentrations of the test substance were analytically verified by GC-MS at the start and the end of the exposure and daily during the exposure of all test concentrations and the control. The measured concentrations of the test substance at the start of the exposure (0 hours) were in the range of 93 to 110% of the nominal values. The measured concentrations of the test substance at the end of the exposure (72 hours) were in the range of lower than the limit of quantification to 42 % of the nominal values. Therefore, all effect values given are based on geometric mean measured test substance concentrations. According to the results of this study, the 72h-ErC50 value was 6.75 mg/L (95% confidence interval: 3.60 – 12.8 mg/L), the 72h-ErC10 value was 5.41 mg/L (95% confidence interval: 2.98 - 6.52 mg/L) and the 72h-NOECr value was 0.326 mg/L. All validity criteria were fulfilled.

Description of key information

EU Method C.3, OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (P. subcapitata) = 6.75 mg/L (95% confidence interval: 3.60 -12.8 mg/L) based on geometric mean measured concentrations.

72h-ErC10 (P. subcapitata) = 5.41 mg/L (95% confidence interval: 2.98 -6.52 mg/L) based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.75 mg/L

EC10 or NOEC for freshwater algae:

5.41 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata in a 72h static condition, according to the EU Method C.3 and the OECD Guideline 201 with GLP statement.

Six concentrations were tested in a geometrical series with a dilution factor of 2 (nominal): 1.60- 3.10- 6.20 - 12.5 - 25.0 - 50.0 mg/L corresponding to geometric mean measured test item concentrations of 0.241 - 0.326- 1.09- 6.54- 16.4- 36.7 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The concentrations of the test substance were analytically verified by GC-MS at the start and the end of the exposure and daily during the exposure of all test concentrations and the control. The measured concentrations of the test substance at the start of the exposure (0 hours) were in the range of 93 to 110% of the nominal values. The measured concentrations of the test substance at the end of the exposure (72 hours) were in the range of lower than the limit of quantification to 42 % of the nominal values. Therefore, all effect values given are based on geometric mean measured test substance concentrations. According to the results of this study, the 72h-ErC50 value was 6.75 mg/L (95% confidence interval: 3.60 – 12.8 mg/L), the 72h-ErC10 value was 5.41 mg/L (95% confidence interval: 2.98 - 6.52 mg/L) and the 72h-NOECr value was 0.326 mg/L, based on growth rate. NOECs are increasingly contested in ecotoxicology because the NOEC is determined as the concentration directly below the LOEC (Lowest Observed Effect Concentration) and depend upon the choice made by the Study Director and not a statistically interpolated value. So, NOEC may occur at concentrations considerably lower than the point at which no biological effect occurs. To use a less selective method, the ECx approach was taken into account. EC10 value was considered to be the most conservative statistical estimation and was chosen as key value, with the EC50 result.