3-(p-methoxyphenyl)-2-methylpropionaldehyde

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:

21 Feb 2016 to 31 Mar 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:

March, 2006; Annex 5 corrected 28 July 2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control at the test start, after 48 and 72 hours. Samples were taken from one replicate of each test concentration. Additionally, reserve samples of 3.0 mL were taken for possible analysis. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Weighing of test item and preparation of test solutions was performed under dimmed light. Preparation of test solutions started with a nominal concentration of 100 mg/L applying one day of magnetic stirring in a closed vessel to accelerate the dissolving of the test item in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/L concentration in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.

ACCLIMATION
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24°C.
- Stock culture medium: M1
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test (see 'Details on test conditions'). The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L (as CaCO3)

Test temperature:

22 - 23 °C

pH:

- Start: 7.3
- End: 7.4 - 7.6

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L (based on a range-finding study)
- Measured concentrations (TWA): 0 (control), 0.24, 0.92, 3.8, 15 and 53 mg/L
- Measured concentrations (initial: t=0h): 0 (control), 0.84, 2.7, 6.9, 19 and 56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
- Type: closed
- Headspace, fill volume: no headspace, fill volume 120 mL
- Aeration: no
- Replicates: 3 replicates of each test concentration; 6 replicates of the control; 1 extra replicate of the control and all test concentrations for sampling purposes; 1 or 2 replicates of each test concentration without algae.
- Initial cells density: 1E+04 cells per mL

GROWTH MEDIUM
- Medium: Adjusted M2. Standard M2 according to the OECD 201 Guideline but with a larger amount of NaHCO3, the addition of HEPES buffer and a lower pH. These adjustments were made because of the use of a sealed exposure system which may result in culture growth being limited by CO2 depletion and increasing pH. Thus additional sodium bicarbonate was added to the medium to provide a source of carbon dioxide for algal growth, an initial lower pH used and buffer added to minimise pH change. This medium was formulated using Milli-RO water and had 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 300 mg/L; HEPES buffer 6 mmol/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L); pH 7.1 ± 0.3.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Pre-culture medium same as test medium
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature of medium was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 88 µE.m-2.s-1.
- Incubation Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. At 24, 48 and 72 hours, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the control, the 10 and 32 mg/L concentration to observe for any abnormal appearance of the algae.

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations: 0 (control), 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Inhibition of algal growth >50% was observed only at the highest test concentration. The expected EC50 for inhibition of both growth rate and yield was between nominal concentrations of 10 and 100 mg/L. Samples taken from nominally 0.10 and 100 mg/L were analysed. The analytical results showed that at a nominal concentration of 100 mg/L the initial concentration was only 63% of nominal. This concentration remained stable during the 72-hour test period (97% of initial at the end of the test). The lowest nominal concentration of 0.10 mg/L was in agreement with nominal at the start and was still in agreement with nominal after 24 hours of exposure (84% of initial). However, the test concentration decreased significantly during the remainder of the test period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 18 - 26 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 3.8 - 8.6 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.L.: 6.1 - 8.3 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.L.: 1.3 - 2.5 mg/L

Details on results:

Growth rates were in the range of the control at TWA concentrations up to and including 0.92 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 3.8 mg/L and higher were increasingly inhibited. Statistically significant inhibition of growth rate was found at TWA concentrations of 3.8 mg/L and higher. See 'Any other information on results incl. tables'. Inhibition of yield increased with increasing test substance concentration from 0.24 mg/L upwards resulting in 99% inhibition at a TWA concentration of 53 mg/L. Statistically significant inhibition of yield was found at TWA concentrations of 3.8 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to TWA concentrations of 15 and 53 mg/L when compared to the control.

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 0.96 mg/L with a 95% confidence interval ranging from 0.95 to 0.97 mg/L. The historical values for growth rate inhibition using standard ‘open’ systems range between 0.82 and 2.6 mg/L.

Reported statistics and error estimates:

The evaluation of the effects was based on the Time Weighted Average (TWA) measured exposure concentrations. For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA and initial concentrations of the test item. The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table: Percentage inhibition of growth rate (total test period) during the final test

TWA concentration test substance (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.655	0.0491	6
0.24	1.617	0.0094	3	2.3
0.92	1.613	0.0296	3	2.5
3.8	1.568	0.0318	3	5.2*
15	1.082	0.3053	3	34.6*
53	0.27	0.0348	3	83.7*

* - effect was statistically significant

Table: Percentage inhibition of growth rate at different time intervals during the final test

TWA concentration test substance (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.898	0	1.527	0	1.54	0
0.24	3	1.733	8.7	1.577	-3.3	1.542	-0.1
0.92	3	1.628	14.2	1.649	-8	1.563	-1.5
3.8	3	1.692	10.8	1.468	3.9	1.544	-0.3
15	3	1.3	31.5	0.993	35	0.954	38
53	3	0.35	81.6	0.517	66.1	-0.056	103.6

 

Table: Percentage inhibition of yield during the final test

TWA concentration test substance (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	143.6	21.33	6
0.24	127.1	3.58	3	11.5
0.92	125.8	11.19	3	12.4
3.8	109.8	10.84	3	23.5*
15	31.1	20.46	3	78.3*
53	1.3	0.23	3	99.1*

* - effect was statistically significant

EFFECT PARAMETERS

Additionally the following effect parameters based on TWA concentrations were derived:

The No Observed Effect Concentrations based on growth rate and yield were 0.92 and 0.92 mg/L, respectively. The ErC20 and EyC20 based on TWA concentrations were 9.6 mg/L (95% C.L.: 6.7 - 12 mg/L) and 3.0 mg/L (95% C.L.: 2.3 - 3.7 mg/L), respectively.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl. tables'.

Conclusions:

The 72-h ErC50 and 72-h ErC10 are 21 mg/L and 6.3 mg/L, respectively, in green algae (Pseudokirchneriella subcapitata).

Executive summary:

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions. Test concentrations were analytically verified at t=0h (start), t=24h and t=72h (end). The analytical results showed that at a nominal concentration of 100 mg/L the initial concentration was only 56% of nominal. This concentration remained stable during the 72-hour test period (88% of initial at the end of the test). All lower test concentrations decreased during the test period, either from the start or after 24 hours of exposure. Therefore, all results are based on the Time Weighted Average (TWA) exposure concentrations which were calculated to be 0 (control), 0.24, 0.92, 3.8, 15 and 53 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the algae were inspected microscopically for abnormalities. At the end of the test the percentage inhibition of yield and average specific growth rate were calculated. No abnormal cells were observed and the cell numbers recorded indicate a clear concentration-response relationship regarding inhibition. Growth rate of algae exposed to 3.8 mg/L and higher were increasingly inhibited. Based on these findings, the 72-h EC50 values for growth rate and yield were determined at 21 mg/L and 7.1 mg/L, respectively. The 72-h EC10 for growth rate and yield were determined at 6.3 mg/L and 2.0 mg/L, respectively.

Description of key information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions. Test concentrations were analytically verified at t=0h (start), t=24h and t=72h (end). The analytical results showed that at a nominal concentration of 100 mg/L the initial concentration was only 56% of nominal. This concentration remained stable during the 72-hour test period (88% of initial at the end of the test). All lower test concentrations decreased during the test period, either from the start or after 24 hours of exposure. Therefore, all results are based on the Time Weighted Average (TWA) exposure concentrations which were calculated to be 0 (control), 0.24, 0.92, 3.8, 15 and 53 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the algae were inspected microscopically for abnormalities. At the end of the test the percentage inhibition of yield and average specific growth rate were calculated. No abnormal cells were observed and the cell numbers recorded indicate a clear concentration-response relationship regarding inhibition. Growth rate of algae exposed to 3.8 mg/L and higher were increasingly inhibited. Based on these findings, the 72-h EC50 values for growth rate and yield were determined at 21 mg/L and 7.1 mg/L, respectively. The 72-h EC10 for growth rate and yield were determined at 6.3 mg/L and 2.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

21 mg/L

EC10 or NOEC for freshwater algae:

6.3 mg/L