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Ecotoxicological information

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:
From 03 Feb 2017 to 29 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH-value in the controls increased by a maximum of 2 units. This deviation has no influence on the integrity of the study results, since all cell-growth related validity criteria were fulfilled.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-ethoxy-4-hydroxybenzaldehyde
EC Number:
204-464-7
EC Name:
3-ethoxy-4-hydroxybenzaldehyde
Cas Number:
121-32-4
Molecular formula:
C9H10O3
IUPAC Name:
3-ethoxy-4-hydroxybenzaldehyde
Test material form:
solid: particulate/powder
Details on test material:
see confidential details
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Solubility in water: 2.82 g/L (25 °C)
- pH: 4.3 (1 % (m/v)) Aqueous solution (pKa: -5.0 – 7.8)
- Vapour pressure: 0.0003 hPa (25 °C)
Method: OECD Test Guideline 104
- Specific density: 1.307 g/cm3 (20 °C) Method: OECD Test Guideline 109
Relative density: 1.307 (20 °C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of stock and/or test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded. Samples were transferred to the test site for chemical analysis under the required storage conditions.

Table: Sampling schedule for chemical analysis during the test period.
Test Period S Control C1 C2 C3 C4 C5 C6
At the start of exposure R XR XR XR XR XR* XR XR
At the end of exposure with algae XR XR XR XR XR XR XR
At the end of exposure without algae – – – – – – – RR

S Stock solution
C1–6 Test solutions
X Sample to be analysed
R Stabilised sample kept as reserve

* The result for the sample taken at start of exposure exceeded the expected nominal concentration by a factor of 10. This sample was therefore re-analyzed, which confirmed the initial result. Therefore, the respective duplicate sample was analysed. The concentration measured in the duplicate sample (C4, day 0) matched the nominal concentration. The result for the initial sample was therefore regarded to be due to an error during sampling or labelling, and was not used for further calculations.















Test solutions

Vehicle:
no
Details on test solutions:
A stock solution (S1) was prepared on the day of the test start by diluting 0.1003 g of the test item in 1000 mL of growth medium, resulting in a nominal concentration of 100 mg test item/L.
The stock solution (S1) was stirred for 10 minutes using a magnetic stirrer, manually shaken overhead, stirred for another 10 minutes, manually shaken overhead again, and then stirred over 50 minutes. The test solutions were then prepared by diluting the stock solution S1 with culture medium.
The volume of the stock solution S1 was large enough to prepare all replicates of the test concentrations and all analytical samples at once.
The following table describes the procedure for preparing the test solutions, and the control for the test.

Table: Preparation of test solutions (control and concentration levels C1–C5).

Code of Test solutions Nominal concentration [mg test item/L] Total volume of test solutions [mL] Code of application/ stock solution Volume of application/ stock solution [mL] Volume of algal preculture [mL]
C0 0.0 1000 – – 7.813
C6* 100 500 C6 500 3.906
C5 30.0 500 C6 150 3.906
C4 10.0 500 C6 50 3.906
C3 3.00 500 C6 15 3.906
C2 1.00 500 C6 5 3.906
C1 0.300 500 C6 1.5 3.906

* 500 mL of S1 (=C6) were transferred to a 500 mL-volumetric flask and 3.906 mL of the pre-culture of Raphidocelis subcapitata were added, resulting in an algal cell concentration of approximately 0.5×104 cells/mL. 200 mL of S1 (=C6) were used without algae for stability purposes.

After temperature adaptation of the test solutions, the required volumes of the algal pre-culture (Raphidocelis subcapitata) were added to each of the volumetric flasks containing 500– 1000 mL of the test solutions to achieve an algal cell concentration of approximately 0.5×104 cells/mL. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 3 times overhead, before adding 100±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells.
The test vessels were placed onto a shaker under light- and temperature-controlled conditions.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The unicellular alga Raphidocelis subcapitata was chosen as a representative of freshwater algae. The selection of the test system was based on the test guideline.

TEST ORGANISM
- Strain: Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata) (SAG 61.81)
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany

ACCLIMATION
- Acclimation period: yes, To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
- Culturing media and conditions (same as test or not): Mod. OECD medium (OECD 201, EN ISO 8692)
pH-value of the algal medium: 8.1
Amount of liquid stock culture per pre-culture vessel: 100±5 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of replicates: 2
Light: Permanent illumination (24/0 h light/dark); fluorescent tubes of universal white type
Light intensity: 60–120 μE m–2 s–1
Shaker: 100±5 oscillations/min
Temperature in the test room: 21–24°C, controlled at ±2°C


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
Temperature in the test media was measured automatically in one test vessel during the period of the test:
Minimum value: 21.70 °C
Maximum value: 21.90 °C
Mean value: 21.85 °C
Number of measurements: 77
The temperature was within the required range of 21–24°C, controlled at ±2°C, as required in the guideline and the study plan.
pH:
pH of untreated test medium: 7.6–9.6
pH in test solutions (exposure): 7.2–9.7
The pH-value in the controls increased by a maximum of 2 units. Therefore, the pH is outside the required range (pH should not vary by more than 1.5 units) as given in the guideline and the study plan. This deviation has no influence on the integrity of the study results, since all cell-growth related validity criteria were fulfilled.
Nominal and measured concentrations:
definitive test:
Nominal: 0, 0.3, 1.0, 3.0, 10, 30 and 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks , Volume of test solution per test vessel: 100±5 mL
- Age of the pre-culture: 3 days
- Number of cells per mL in the pre-culture before inoculating the test solution: 64×104
- Number of cells per mL test solution at the beginning of the test: 0.5×104
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Number of replicates for stability check (highest concentration, without algae): 2


TEST MEDIUM / GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition:
The growth medium was OECD medium as described in the test guideline, modified as described below. The preparation of the synthetic algal medium is described in detail in ECT Standard Operating Procedure (SOP) A 6.1. To optimise growth of Raphidocelis subcapitata, however, the OECD growth medium was prepared with a 10-fold concentrated iron (Fe)-EDTA complex compared to the original recipe according to guideline OECD 201. The growth rates of the algae in pre-culture were not sufficient in original OECD medium but satisfactory in the modified medium, which is fully described below.
NaHCO3 50.0 mg/l
NH4Cl 15.0 mg/l
MgCl2 × 6 H2O 12.0 mg/l
CaCl2 × 2 H2O 18.0 mg/l
MgSO4 × 7 H2O 15.0 mg/l
KH2PO4 1.6 mg/l
FeCl3 × 6 H2O 0.640 mg/l
Na2EDTA × 2 H2O 1.000 mg/l
H3BO3 0.1850 mg/l
MnCl2 × 4 H2O 0.4150 mg/l
ZnCl2 0.0030 mg/l
CoCl2 × 6 H2O 0.0015 mg/l
Na2MoO4 × 2 H2O 0.0070 mg/l
CuCl2 × 2 H2O 0.00001 mg/l

Justification for modification of growth medium: The steady and sufficient supply of trace elements (e.g. iron) is essential for algal growth. In this context, the introduction of chelating agents (e.g. EDTA) is very valuable, since by using these compounds, trace element cations easily form reversible complexes, whose splitting steadily supplies the algae with the amounts required for growth. There was no indication of interaction between this medium and the test item.
Before use, the pH of the medium was adjusted to 8.1±0.2. The algal medium was sterilised (filtered, pore size 0.2 μm) before use. The vessels used were sterilised using a drying oven for three hours at 150 °C.

WATER PARAMETERS
After temperature adaptation of the test solutions, the pH was measured and recorded for each test concentration and the control in one replicate. Temperature was recorded once per hour throughout the test in a separate test vessel placed
on the shaker containing approx. 100 mL deionised water. At the end of the test, the pH was measured and recorded in pooled replicates of each concentration and the control.

OTHER TEST CONDITIONS
Light regime: 24 h light/0 h dark
Type of light: Fluorescent tubes of universal white type (Osram Lumilux L 58W/865)
Light intensity: Mean 86.9 μE m–2s–1
Shaker: 100±5 oscillations/min; the test vessels were placed randomly on the shaker


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). Morphological deviations compared to the control cells were recorded.
After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 μL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results [RFU, relative fluorescence units] were converted into biomass concentration [cells/mL] using a calibration line (Figure 1); calculated with StatSoft, Inc. (2013). STATISTICA (data analysis software system), version 12. A dilution series of the pre-culture was used to determine the calibration line by comparison of cell numbers (counted using a microscope) and fluorescence intensity (measured using a fluorometer) in consideration of background fluorescence of the blank algal medium.
- Other:
Endpoints: ECx (e.g. EC50, EC20, EC10), NOEC/LOEC
Biological parameters: Inhibition of growth in relation to control (growth rate & yield)
NOEC, LOEC and ECx values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and nominal concentrations of the test item (mg/L).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.8, 4, 20, 100 mg/L
- Results used to determine the conditions for the definitive study:
Growth Rate of Raphidocelis subcapitata
%Inhibition caused by the test item after 72 h.
mg/L % inhibition
Control 0.0
0.80 1.5
4.00 5.3
20.00 5.2
100.00 21.1






Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
not determinable
Remarks:
beyond tested concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
21.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
not determinable
Remarks:
beyond tested concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
30 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
The factor of biomass increase in the control replicates was 227 within the 72-hour exposure period. Therefore, the data were in the required range (factor of at least 16) as given in the guideline and the study plan.
- Observations: In the control (C0) and up to concentration level C6 (100 mg test item/L) no deformed and/or damaged algal cells were observed during microscopic inspection at the end of the test.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- The most recent reference test with potassium dichromate (K2Cr2O7) as reference item performed in a separate study (Study No. IAO1610) showed the following result:
Result: Growth Rate ErC50 (95%-CL): 0.801 (0.747 – 0.855) mg/L
Based on an international ring test (ISO (2004). Water Quality – Freshwater algal growth inhibition test with unicellular green algae. European Standard EN ISO 8692, October 2004.) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories was 1.19 mg/L with a standard deviation of 0.27 mg/L. The EμC50 value for the toxic reference item, potassium dichromate, was determined as 0.801 mg/L. Although this value is slightly outside the range reported in the ring test mentioned above, this value is within the historical range of the general reference test results of our laboratory (mean EC50 value = 1.079 ± 0.361 mg/L) and within the narrow upper (1.447 mg/L) and lower (0.718 mg/L) warning limits. The narrow warning limits were calculated as one standard deviation from the historical mean of the EμC50 values. The calculation of the warning limits was not conducted in compliance with Good Laboratory Practice Principles (all calculated EμC50 using the log concentration; according to Environment Canada (2005), Guidance Document on Statistical Methods for Environmental Toxicity Tests, Method Development and Applications Section, Environment Canada, Ottawa, ON, Report EPS 1/RM/46). Furthermore,
the EμC50 value is within the refined range of 1.10 ± 0.48 mg/L, recommended based on an international ring test (Pattard, M.; Römbke, J.; Moser, T. (2009). Range of Reference Tests in Aquatic Tests. [in] Moser, H.; Römbke, J. “Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test”, chapter 5, pp. 61-70.). Therefore the results of this reference test are acceptable and the test conditions are reliable.
Reported statistics and error estimates:
The statistical analysis was performed using the nominal concentrations of the test item. The data were evaluated by the Shapiro-Wilk's Test for normal distribution, and by Levene's Test for homogeneity of variances. The Williams Multiple Sequential t-test for homogenous variances was applied to find out whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentrations. The significance level was 0.05.
Weibull analysis using linear maximum likelihood regression was used to calculate the ECx values as based on nominal test concentrations in mg test item/L. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg
15, D-52477 Alsdorf) was used for these calculations.

Any other information on results incl. tables

Analytical Results

Samples from the test solutions were analysed using an HPLC-MS/MS method to determine actual levels of the test item in comparison to nominal test concentrations.

Table n°1: Summary of measured concentrations at the beginning and at the end of the test.

Conc. Level

Test period (d)

Nominal concentration (mg test item/L)

Measured concentration (mg test item/L)

Recovery of nominal (%)

Geometric mean measured concentration over 72 h (mg test item/L)

Control

C1

C2

C3

C4

C5

C6

0

0

0

0

0

0

0

0

0.3

1

3

10

30

100

< LOD

0.262

0.909

2.92

10.91

26.9

85.8

-

87.3

90.9

97.3

109

89.7

85.8

 

Control

C1

C2

C3

C4

C5

C6

3

3

3

3

3

3

3

0

0.3

1

3

10

30

100

< LOD

0.0225*

0.0225*

0.0225*

6.84

16.7

69.3

-

n.d.

n.d.

n.d.

68.4

55.7

69.3

n.d.

0.0768

0.143

0.256

8.64

21.2

77.1

LOQ = Limit of Quantification (0.15 mg/L)

n.d.: not determined, LOD = Limit of Detection (0.045 mg/L)

*actually measured value <LOD; replaced by 50% of LOD (0.0225 mg/L) for calculation of geometric mean concentration

Table n°2: Summary of biological results [mg test item/L] based on nominal concentrations

Yield

EyC10

EyC20

EyC50

NOEC

LOEC

Nominal concentration (mg test item/L)

Lower 95%-cl

Upper 95%-cl

45.2

 

20.8

58.8

61.6

 

38.4

72.6

98.3

 

91.2

105.7

30.0

100

 

Growth rate

ErC10

ErC20

ErC50

NOEC

LOEC

Nominal concentration (mg test item/L)

Lower 95%-cl

Upper 95%-cl

88.9

 

79.1

93.1

Beyond tested concentrations

Beyond tested concentrations

30.0

100

cl: confidence limit

Table n°3: Summary of biological results [mg test item/L] based on measured test item concentrations.

Yield

EyC10

EyC20

EyC50

NOEC

LOEC

Measured concentration (mg test item/L)

Lower 95%-cl

Upper 95%-cl

32.2

 

 

14.9

42.6

45.3

 

 

28.3

53.9

75.7

 

 

69.8

82.0

21.2

77.1

 

Growth rate

ErC10

ErC20

ErC50

NOEC

LOEC

Measured concentration (mg test item/L)

Lower 95%-cl

Upper 95%-cl

67.8

 

 

61.8

70.9

Beyond tested concentrations

Beyond tested concentrations

21.2

77.1

cl: confidence limit

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Mean biomass increase in the control cult.: 227 Mean coef. of variation for section-by-section specific growth rates in the control cult.: 15.3% Coef. of variation of average specific growth rates during test period in replicate control cult.: 1.4%
Conclusions:
Ethylvanillin is considered as not harmful for aquatic organisms. (EC50, 72h > 100 mg/L).
Executive summary:

In a 72 hours algae study (ECT, 2017), the toxicity of the test item 3-ethoxy-4-hydroxybenzaldehyde (ethylvanillin) was evaluated in the algal strain Pseudokirchneriella supcapitata using a 72-hour static test according to current EC and OECD guidelines, and GLP. Based on measured concentrations the results of the study were:

- The 72-hour ErC50 (geometric mean) was > 100 mg/L.

- The 72-hour EyC50 (geometric mean) was 75.7 mg/L.

- The NOEC was 21.2 mg/L for both yield and growth rate.

The validity criteria were fulfilled.

In this study, ethylvanillin is considered as not harmful to algae.