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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-21 to 2012-07-05
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
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test material was described with the generic name "Cyprinal", chemical name "alpha-methylcinnamaldehyde" and described with the generic CAS 101-39-3/EC 202-938-8. As manufactured by Innospec, the product met the criteria of > 80% trans-isomer, supporting that the test material was the isomer better described as CAS 15174-47-7.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Innospec Limited; Batch no. A101112A
- Expiration date of the lot/batch: 2012-11-12
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C ± 3°C and N2 atmosphere (headspace), (tids)
- Stability under test conditions: Stable under normal conditions of storage and use, hazardous polymerization will not occur.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Yellow clear liquid

OTHER SPECIFICS:

Purity: >99%
Solubility in solvents: easily soluble in methanol and acetone
Volatility: 3.33 Pa at 25°C
Water Solubility: 490 mg/L
Density: 1.036-1.040 at 20°C
Analytical monitoring:
yes
Details on sampling:
At the start (0h), after 24h, after 48h and at the end of the experimental phase (72h) samples (4.6 ml) were taken from the highest concentration, the middle concentration, the lowest concentration and the control for chemical analysis. At the end of the study additional samples were taken from the test vessels with algae. The algae were separated by filtration (0.45 µm CA filter) to avoid disturbance during the analytical process.

The samples were taken in duplicate. Two additional vessels were created for analytical purposes and from these 5 ml were taken at each sampling point (0h, 24h, 48h and 72h) and mixed in 10 ml glass beakers. 3.8 ml were transferred into brown glass vials with screw cap. The test item was kept under inert gas atmosphere, amd the head spaces in the glass vials were overlaid with N2 atmosphere. Immediately after sampling the vials were stored in a refrigerator at 5°C ± 3°K for a maximum period of 8 days. Then they were sent by over-night express service to the analytical laboratory. Transport required less than 24 hours and was performed in an isolated box containing cooling elements. The analytical laboratory stated that the samples arrived in good condition.

The min/max temperature in a reference flask was recorded on a daily basis. During the time of exposure the temperature was in the range of 23.0 to 23.3°C. At the start of the test {Oh) and after 24, 48 and 72 h the biomass was determined by measuring the chlorophyll fluorescence for each test vessel. At the end of the test, the pH was measured in one flask from each concentration. The algae cells have been examined microscopically in one replicate from the control and test vessels.
Vehicle:
yes
Remarks:
algal growth medium
Details on test solutions:
PREPARATION OF TEST AND CONTROL VESSELS
A stock solution with a nominal concentration of 111 mg/L (water solubility 490 mg/L) was prepared by adding 54.4 µL (corresponding to 55.5 mg) Cyprinal to 500.6 mL algal medium according to OECD 201 and stirring over a period of 1 hour with a magnetic stirrer. The stock solution was clear and colourless. It was diluted with algal medium to obtain 5 test concentrations intended differing by a factor of 2 as shown in Tables 1a and 1b in "Any other information on methods and materials incl. tables" below.

The concentrations of the test solutions were chosen 1.11-fold higher than the nominal concentrations intended in the test so as to take into account the dilution by the algal inoculum suspension (10%). 45 mL test solution was transferred into each test vessel (100 mL wide necked Erlenmeyer Flask) and 5 mL of the algae inoculum suspension (0.5 x 10^5 algae mL^-1) were added to obtain an algae starting concentration of 0.5 x 10^4 algae/mL. For the controls, 5 mL algae inoculum suspension was added to the 45 mL algal medium. Three replicates have been prepared for the test vessels and six replicates for the control vessels.

For analytical purpose, additional vessels without algae were prepared. These vessels were prepared in the same way as described above with the only deviation that algal medium instead of algal inoculum was added.

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae (Desmodesmus subspicatus)
- Strain: CHODAT (SAG No. 86.81)
- Source (laboratory, culture collection): Culture Collection of Algae (SAG) at the University of Gottingen
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: cultivated in suspension culture

ACCLIMATION
- Acclimation period: Not specified
- Culturing media and conditions (same as test or not): Same
Test type:
static
Water media type:
other: deionised water
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23-23.3°C
pH:
7.4-8.0 (0-72h)
Nominal and measured concentrations:
Nominal Concentrations: 0. 6.25, 12.5, 25, 50, and 100 mg/L
Measured Geometric mean concentrations : 0, 0.1 (measured), 0.36 (estimated), 0.62 (measured), 49.1 (estimated) and 97.6 mg/l (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer Flasks, wide necked
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL, Glass
- Aeration: Not specified
- Initial cells density: 0.5 x 10^5 algal cells/mL
- Control end cells density: 0.5 x 10^5 algal cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Algal Growth Medium according to OECD Guideline 201)
- Detailed composition if non-standard medium was used:

Solution A1
Ammonium Chloride p.a. NH4Cl 1.5g
Magneisum Chloride p.a. MgCl2*6H2O 1.2g
Calcium Chloride dihydrate e.p. CaCl2*2H2O 1.8g
Magnesium Sulphate Heptahydrate p.a. MgSO4.*7H2O 1.5g
Potassium Dihydrogen Phosphate p.a. KH2PO4 0.16g

Solution A2
Iron (III) Chloride Hexahydrate p.a. Fe(III)Cl3*6H2O 0.064g
Sodium-EDTA Dihydrate Na2EDTA*2H2O 0.100g
dissolved in 100 mL deionised water

Solution A3
Boric Acid p.a. H3BO3 0.185g
Manganese Chloride Tetradhydrate p.a. Mn(Cl2)*4H2O 0.415g
Sodium Molybdate Dihydrate p.a. Na2MoO4*2H2O 0.007g
Zinc Chloride p.a. ZnCl2 0.003g
Cobalt Chloride Hexahydrate p.a. CoCl2.*6H2O 0.0015g
Copper Chloride Dihydrate p.a. CuCl2*2H2O 0.00001g
dissolved in 1000 mL deionised water (for ZnCl2, CoCl2 and CuCl2 more concentrated solutions were prepared and the required volumes used for A3)

Solution A4
Sodium Hydrogen Carbonate p.a. NaHCO3 50g
dissolved in 1000 mL deionised water

Solutions A1-A3 were autoclaved, Solution A4 was steriley filtered (membrane pore diameter 0.22 µm)

10-x Concentrated Medium: 100 mL from stock solution A1 and 10 mL from each stock solution A2-A4 were filled up to 1000 mL with deionised water. The medium was aerated for 45 minutes. The pH was 8.5.

1-x concentrated medium was prepared by diluting 100 mL of the 10-x concentrated medium with 900 mL deionised water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: Yes
- Light intensity and quality: Set on 45% of the maximum light intensity resulting in a mean light intensity 120 µE/m^2 s ± 5.7% (PAR, measured by a spherical sensor)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Counter Z2
- Chlorophyll measurement: Chlorophyll fluorescence measured at an excitation wavelength of 465 nm and an emission wave length of 670 nm using a fluorescence micro-plate reader.

Positive control
A quality check takes place at regular intervals using a concentration range of potassium dichromate (Sigma, Steinheim, Germany, Lot No. 074K0617, from November, 22"•, 2005) in order to assess the validity of the test system. The last quality check was performed in May 2012.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.12 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.:
0.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
With the highest two concentrations tested of 100 mg/L and 50 mg/L (nominal) algal growth rate was completely inhibited. With the next three lower concentrations growth inhibition decreased dose dependent. The following ECx values were calculated with the nominal concentrations:

Growth Rate (nominal Concentrations):
1) ErC50 = 14.8 mg/L (95% CI: 12.8 - 17.0 mg/L)
2) ErC20 = 8.2 mg/L (95% CI: 6.3 - 9.8 mg/L)
3) ErC10 = 6.1 mg/L (95% CI: 4.2 - 7.6 mg/L)

Yield (nominal concentrations):
1) EbC50 = 7.8 mg/L (95% CI: 7.7 - 7.9 mg/L)
2) EbC20 = 4.5 mg/L (95% CI: 4.4 - 4.6 mg/L)
3) EbC10 = 3.4 mg/L (95% CI: 3.3 - 3.5 mg/L)

For Growth Rate and Yield: NOEC <6.25 mg/L and LOEC ≤ 6.25 mg/L

During the test, Cyprinal was stable only in the highest concentration. With the middle concentration (25 mg/L) after 48 hours Cyprinal concentration was <0.10 mg/L (LLOQ). With the lowest concentration (6.25 mg//L) Cyprinal had already disappeared after 24 hours.

The concentrations analysed were below 80% of the nominal concentrations for several testunits. Therefore the geometric mean concentrations of the measured values at 0h, 24h, 48h and 72h were calculated according to the formula given in Annex 2 of the OECD Guidance Document No. 23 "on Aquatic Toxicity Testing of difficult Substances and Mixtures". For the test concentrations not analysed (C: 50 mg/l and E: 12.5 mg/l) the mean between the next higher and the next lower concentration was calculated.

The ECx values were were therefore calculated with the geometric mean exposure concentrations.
Growth Rate (Geometric mean measured concentrations):
1) ErC50 = 0.39 mg/L
2) ErC20 = 0.18 mg/L
3) ErC10 = 0.12 mg/L

Yield (Geometric mean measured concentrations):
1) EbC50 = 0.13 mg/L
2) EbC20 = 0.05 mg/L
3) EbC10 = 0.03 mg/L

It can be observed that the cell count in the highest test concentration (A: 100 mg/L) decreases already during the first 24 hours and considerably between 24 hours and 48 hours of the exposure period. With the next lower concentration (B: 50 mg/L) cell count decreases especially during the period between 24 and 48 hours. These observations might be attributed to the toxic effect of Cyprinal. With the next lower concentration (C: 12.5 mg/L) it can be observed that the cell count decreases during the first 48 hours and then cell count increases again. This coincides with the decrease of Cyprinal in the test solution as demonstrated for the measured concentrations. With the decrease of the Cyprinal concentration during the test course toxicity disappears also.

It can be stated that the loss of substance occurs only for the conditions of exposure. A stability test performed by the analytical laboratory showed that the samples themselves were stable over a period of six days at 5° ± 3°C. Deviation between the measured data at the beginning and at the end of the six day's storage period was 0.9%.

The calculation of the geometric mean concentration has to be regarded with caution because with the loss of substance toxicity disappears also. For the calculation of the geometric mean the measured concentrations
Criteria for Validity of the Test (after 72 hours of exposure):

1) The biomass in the controls increased within 72 hours from 0.05 x 10^5 algal cells/mL to 3.37 x 10^5 (factor 67) which is within the range for validity of the test (required ≥16).
2) The maximum mean variation co-efficient of the growth rate from day to day (day 0-1; 1-2; and 2-3) was 20.0% in the controls which is within the validity range of 0-35%.
3) The variation co-efficient calculated for the mean growth rate from day 0 to day 3 was 3.1% and below the upper limit of 7%.

The Test was valid according to OECD 201.
Results with reference substance (positive control):
A quality check takes place at regular intervals using a concentration range of potassium dichromate (Sigma, Steinheim, Germany, Lot No. 074K0617, from November, 22nd, 2005) in order to assess the validity of the test system. The last quality check was performed in May 2012. The EC50 (growth rate) was 0.76 mg/I (0.68 - 0.85 mg/L 95%-CI) and the EyC50 (yield) was 0.42 mg/I (0.40 - 0.44 mg/I 95%-CI).

Table 2. Mean Growth Rates and Mean Yields and its Inhibition Compared to the Control

 

Control

A

B

C

D

E

Test Concentration (mg/L)

0

100

50

25

12.5

6.25

Number of Replicates

6

3

3

3

3

3

Mean Growth Rate/d

1.375

0

0

0.315

0.847

1.182

Mean Yield**

3018

0

0

100

707

1908

Mean Inhibition of Growth Rate

 

100%

100%

77.13%

38.4%

14.0%

Mean Inhibition of Yield

 

100%

100%

96.7%

76.6%

36.8%

** Chlorophyll Fluorescence Units

 

Table 3. Parameters of the Dose-Response Curves (inhibition of specific growth rate and yield)

 

Specific Growth Rate

72-hour Nominal Concentration

Yield

72-hour Nominal Concentration

EC50

95% CI

14.8 mg/L

7.8 mg/L

12.8 – 17.0 mg/L

7.7 – 7.9 mg/L

EC20

95% CI

8.2 mg/L

4.5 mg/L

6.3 – 9.8 mg/L

4.4 – 4.6 mg/L

EC10

95% CI

6.1 mg/L

3.4 mg/L

4.2 – 7.6 mg/L

3.3 – 3.5 mg/L

Slope of the Curve

P(Chi2)

degrees of freedom

r2

3.3029

3.5567

0.9993

1.0000

3

3

0.978

1.000

NOEC

LOEC

<6.25 mg/L

<6.25 mg/L

≤6.25 mg/L

≤6.25 mg/L

95% CI = 95% Confidence Limit

Data were calculated/determined using the statistical software ToxRat (Version 2.09, 03/2004, ToxRat Solutions, Alsdorf, Germany).

For the calculations values of 100% inhibition were set on 99.995%.

Table 4. ECx Values for Measured Concentrations

Parameter

ECx

Value

Growth Rate

ErC50

0.39 mg/L

ErC20

0.18 mg/L

ErC10

0.12 mg/L

LOErC

≤0.1 mg/L

NOErC

<0.1 mg/L

Yield

EyC50

0.13 mg/L

EyC20

0.05 mg/L

EyC10

0.03 mg/L

LOEyC

≤0.1 mg/L

NOEyC

<0.1 mg/L

Validity criteria fulfilled:
yes
Conclusions:
In a study performed according to OECD 201 Algae, Growth Inhibition Test, 2-methyl-3-phenyl-2-propanal was shown to have an EC50 (growth) 0.39 mg/L (geometric mean measured) . The EC10 (growth) was 0.12 mg/L (geometric mean measured).
Executive summary:

In a key algal growth inhibition test conducted according to OECD Guideline 201, the potential of the test material (Cyprinal; CAS# 101-39-3) to inhibit the growth of freshwater green microalgae (Desmodesmus subspicatus) was evaluated.

 

Exponentially growing green algae were exposed to the test material in batch cultures at nominal concentrations of 0, 6.25, 12.5, 25, 50, or 100 mg/L (reported to be geometric mean measured concentrations of 0, 0.1, 0.36, 0.62, 49.1, 97.6 mg/l, respectively) over a period of 72 hours. Growth and inhibition of the algal cultures were quantified from measurements of algal biomass as a function of time.

The reduction of growth in algal cultures exposed to various concentrations of the test material were determined by measuring chlorophyll fluorescence as a measure for algal biomass. Chlorophyll fluorescence was measured in a 96-well micro-plate using a fluorescence micro-plate reader (excitation wave length: 465nm; emission wave length: 670 nm). As required OECD Guideline 201, to determine the conversion factor between the chlorophyll fluorescence and the algal count, a correlation between the two was provided for difference algal cell concentrations using the same algal pre-test culture as in the test. The algal count was determined for a cell size between 3 and 9 µm using a Coulter Counter Z2. Three replicates each were used for the test concentrations while six replicates were used for the control vessels. The specific growth rate and yield for each replicate and the inhibition of the treated algae compared to the controls were calculated after 24, 48, and 72 hours.

 

With the highest two concentrations tested of 100 mg/L and 50 mg/L (nominal) (reported to be mean measured concentrations of 97.6 and 49.1 mg/l, respectively), algal growth rate was completely inhibited. With the next three lower concentrations, growth inhibition decreased dose dependently. As the measured concentrations were found to decrease to <80% of the nominal concentrations over the course of the study the geometric mena measured concentrations have been reported.

 

For the Growth Rate ErC50 the mean measured concentration was 0.39 mg/L and for the the ErC10 mean measured concentration was 0.12 mg/L.

ECxValues for Mean Measured Concentrations

Parameter

ECx

Value

Growth Rate

ErC50

0.39 mg/L

ErC20

0.18 mg/L

ErC10

0.12 mg/L

LOErC

≤0.1 mg/L

NOErC

<0.1 mg/L

Yield

EyC50

0.13 mg/L

EyC20

0.05 mg/L

EyC10

0.03 mg/L

LOEyC

≤0.1 mg/L

NOEyC

<0.1 mg/L

Description of key information

In a key algal growth inhibition test conducted according to OECD Guideline 201, the potential of the test material (Cyprinal; CAS# 101-39-3) to inhibit the growth of freshwater green microalgae (Desmodesmus subspicatus)was evaluated. For the Growth Rate ErC50the mean measured concentration was 0.39 mg/L and for the ErC10 mean measured concentration was 0.12 mg/L.

ECxValues for Mean Measured Concentrations

Parameter

ECx

Value

Growth Rate

ErC50

0.39 mg/L

ErC20

0.18 mg/L

ErC10

0.12 mg/L

LOErC

≤0.1 mg/L

NOErC

<0.1 mg/L

Yield

EyC50

0.13 mg/L

EyC20

0.05 mg/L

EyC10

0.03 mg/L

LOEyC

≤0.1 mg/L

NOEyC

<0.1 mg/L

 

Data from an OECD 201 growth inhibition of algae study on hexylcinnamaldehyde has also been provided to support the read-across of long-term toxicity to daphnia and short-term toxicity to fish data from hexylcinnamaldehyde to (2E)-2 -methyl-3 -phenylacrylaldehyde.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.39 mg/L
EC10 or NOEC for freshwater algae:
0.12 mg/L

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