7-(4-ethyl-1-methyloctyl)quinolin-8-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:

21 - 25 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

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:

adopted 23 March 2006

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 07 December 2015

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Principles of method if other than guideline:

Deviations:
The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved. The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

7-(4-ethyl-1-methyloctyl)quinolin-8-ol

EC Number:

277-531-1

EC Name:

7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Cas Number:

73545-11-6

Molecular formula:

C20H29NO

IUPAC Name:

7-(5-ethylnonan-2-yl)quinolin-8-ol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 1702-18-01/O
- Expiration date of the lot/batch: 25 March 2021
- Purity test date: 26 March 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel dark and dry at 16.2 – 23.7 °C.
- Solubility and stability of the test substance in the solvent/vehicle:
Stability in solvents H2O: unknown; Ethanol: unknown; acetone; unknown; CH3CN: unknown; DMSO: unknown
Solubility in solvents H2O: < 0.1 g/L; Ethanol: unknown; acetone: > 1 g/L; CH3CN: unknown; DMSO: unknown


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
A saturated solution was prepared for the test. This was done by mixing the nominal load of 100 mg/L resp. 100.7 µL/L test item (based on a density of 0.9926 g/mL determined in LAUS study 18081404G912), with the corresponding amount of algal medium (demineral-ised water enriched with minerals but without algae and stirring for 24 hours on a mag-netic stirrer. The test item was pipetted directly onto the surface of the test dilution water. The resulting solution was used unfiltrated.


FORM AS APPLIED IN THE TEST (if different from that of starting material)
The lower treatments (4.6 / 10 / 22 / 46 mg/L nominal) were prepared by dilution of this solution with algal medium.

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For each treatment, 200 mL of the respective test item solution was mixed with the neces-sary amount of algal pre-culture (0.289 mL) to achieve a cell concentration of 2.6 *103 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item so-lution and mixed with the necessary amount of algal pre-culture (0.505 mL). In this mix-ture, the pH-value was measured.
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test the pH value in treatments and blank control was measured again. The content of TOC in the test vessels was measured at the start and at the end of the test.
To avoid adsorption of dissolved test item onto the algae cells, the TOC in the test solutions was measured in extra replicates without algae cells.

- Controls: blank (replicates without algal cells) and potassium dichromate (postive control)

- Other relevant information:
Treatments tested: 4.6 / 10 / 22 / 46 / 100 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests
Number of replicates: 6 replicates for the blank control
3 replicates for each treatment

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: SAG Strain Number: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 96 hr
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.6 – 23.0 °C

Nominal and measured concentrations:

4.6 / 10 / 22 / 46 / 100 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: glass flasks total volume 65 mL
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: 2.6 *103 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each treatment
3 vessels, filled with 45 ±1 mL test solution and algae
1 vessel, filled with 45 ±1 mL test solution without algae for analytical determination
- No. of vessels per control (replicates): 6 replicates for the blank control
6 vessels, each filled with 45 ±1 mL algal medium and algae
1 vessel filled with 45 ±1 mL algal medium without algae for analytical determination
- No. of vessels per vehicle control (replicates): see above

GROWTH MEDIUM
- Standard medium used: yes
All solutions were sterilized before use.


TEST MEDIUM / WATER PARAMETERS
Composition of the Solutions:

Stock Solution I
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL

Stock Solution II
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL


Stock Solution III
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL

Stock Solution IV
NaHCO3 50 g
H2O deionised ad 1000 mL

Algal medium
Stock solution I 10.0 mL
Stock solution II 1.0 mL
Stock solution III 1.0 mL
Stock solution IV 1.0 mL
H2O deionised ad 1000 mL

Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration. The algal medium corresponds to the OECD TG 201 medium.
Deviations from the nominal weighted loads were less than 5 %.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: within the specified range (4440 – 8880 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44.45 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): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Because of the very low solubility of the test item, the measured concentrations in the test solutions were in a similar low range as in the control.
- Effect concentrations exceeding solubility of substance in test medium: yes

Any other information on results incl. tables

Findings

Cell Numbers

The cell numbers were determined with an electronic particle counter, the details are given in the attached study report. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

Table1.Cell Number/mL

Nominal Concentrationin mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2600	7013	47517	265670
Blank control	SD	0	697	8087	13104
4.6	Mean	2600	6820	54173	325353
4.6	SD	0	197	1251	2525
10	Mean	2600	7000	54493	281093
10	SD	0	802	8911	12108
22	Mean	2600	5313	20707	127100
22	SD	0	539	871	989
46	Mean	2600	6227	11087	15280
46	SD	0	462	319	560
100	Mean	2600	4473	9127	8673
100	SD	0	155	809	480

SD = Standard Deviation

pH values

In the following table,the pH values measured at the start and the end of the test are stated:

Table 2. pH values

Nominal Concentrationin mg/L	0 h	72 h
Blank control	7.4	7.6
4.6	7.4	7.9
10	7.5	8.0
22	7.5	7.4
46	7.5	7.3
100	7.5	7.3

 

 

Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated:

Table 3. Microscopical Observations

Nominal Concentrationin mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
4.6	Yes
10	Yes
22	Yes
46	No cells visible
100	No cells visible

 

Analytical Determination

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the total organic carbon (TOC) content in the test solutions using a carbon analyser.

Because of the very low solubility of the test item, the measured concentrations in the test solutions were in a similar low range as in the control.

The details are given in the following tables:

Table 4. Measured Concentrations IC and TC

Nominal Concentration Test Item	Measured TC  t = 0 h	Measured TC  t = 72 h	Measured IC  t = 0 h	Measured IC  t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	7.36	7.12	7.08	6.72
4.6	7.53	7.11	7.18	6.78
10	7.61	7.14	7.10	6.92
22	7.77	7.09	7.21	6.86
46	7.93	8.11	7.20	7.01
100	8.38	8.56	6.95	6.96

LOQ (Limit of quantification) TC = 0.897 mg/L

LOQ (Limit of quantification) IC = 1.59 mg/L

Table 5. Calculated Concentrations TOC

Nominal Concentration Test Item	Calculated TOC (TC-IC)  t = 0 h	Calculated TOC (TC-IC)  t = 72 h
mg/L	mg/L	mg/L
Blank control	0.28	0.40
4.6	0.35	0.32
10	0.51	0.22
22	0.56	0.23
46	0.73	1.10
100	1.43	1.60

 

Growth Rate, Yield

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Table 6. Growth Rate µ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	1.54	263070
	SD	0.02	13104
4.6	Mean	1.61	322753
	SD	0.00	2525
10	Mean	1.56	278493
	SD	0.01	12108
22	Mean	1.30	124500
	SD	0.00	989
46	Mean	0.59	12680
	SD	0.01	560
100	Mean	0.40	6073
	SD	0.02	480

SD = Standard Deviation

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

Table7.Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
4.6	-4.4	-22.7
10	-1.2	-5.9
22	15.9	52.7
46	61.7	95.2
100	74.0	97.7

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

 

Statistics and Results

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in the study report.

Biological Results

The following values were determined:

Table8.Biological Results of the Test Item

Parameter	Value	95 % confidence interval
NOEC (Growth Rate) 72 h	10 mg/L	--
NOEC (Yield) 72 h	10 mg/L	--
LOEC (Growth Rate) 72 h	22 mg/L	--
LOEC (Yield) 72 h	22 mg/L	--
72h ErC10	14.57 mg/L	14.20 – 14.93 mg/L
72h EyC10	13.70 mg/L	11.24 – 16.69 mg/L
72h ErC50	44.45 mg/L	43.93 – 44.99 mg/L
72h EyC50	21.60 mg/L	20.69 – 22.54 mg/L

 

Validity

Increase Factor

The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

Daily Growth Rates

Mean coefficient of variation of daily growth rates in the blank controls should be 35 % at the most.

Coefficient of variation of average growth rate during the whole test period should be 7 % at the most.

Values and assessment

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables.

Table9.Daily Growth Rates of the blank controls

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	0.953	2.249	1.483	42%	1.561
Replicate 2	0.863	1.975	1.815	39%	1.551
Replicate 3	1.024	1.844	1.778	29%	1.549
Replicate 4	1.096	1.757	1.789	25%	1.547
Replicate 5	0.898	1.919	1.757	36%	1.524
Replicate 6	1.096	1.699	1.761	24%	1.519
Mean	0.988	1.907	1.731	33%	1.542
Standard deviation	0.100	0.195	0.123		0.017
CV	10%	10%	7%		1%

CV = Coefficient of Variation

 

Table10.Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	102	Valid
Mean coefficient of variation of daily growth rates	max. 35 %	33%	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7 %	1%	Valid

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L (nominal). Significant inhibition of algal growth was observed at the 3 highest concentrations 22 mg/L, 46 mg/L and 100 mg/L.
At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the total organic carbon (TOC) content in the test solutions using a carbon analyser.
Because of the very low solubility of the test item, the measured concentrations in the test solutions were in a similar low range as in the control.
Therefore, calculation of a mean exposure concentration of dissolved test item was not possible, and the determination of the biological results was based on the nominal concentration (OECD Guidance Doc. No.23, §176).
However, clear toxicity in the treatments showed the presence of dissolved test item in the test solutions.