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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effects of the test item on the growth and physiological characteristics of Chlorella pyrenoidosa were studied.
GLP compliance:
not specified
Remarks:
It concerns a publication. No information is available on the GLP compliance status.
Analytical monitoring:
not specified
Remarks:
It concerns a literature publication. The GLP compliance is not indicated in the article.
Details on sampling:
-Concentrations (nominal): 2.0, 4.0, 8.0 and 10 mg/L
-Sampling: The algae cell concentrations were determined at 0 h, 24 h, 48 h, 72 h and 96 h by particle cell meter with microscopy and photometric measurement using a spectrophotometer at λ 680 nm.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a stock solution was prepared, test concentrations were prepared from the stock solution in Selenite Enrichment (SE) medium.
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): maximum 0.2%
Test organisms (species):
Chlorella pyrenoidosa
Details on test organisms:
TEST ORGANISM
- Source: Chinese Academy of sciences, institute of hydrobiology Wuhan.

ACCLIMATION
-Before initiation of test: algae were cultured in SE medium in Erlenmeyer flasks.
-Temperature: 25 °C
-Light period: 14/10 h (light/dark)
-Light intensity: 3000-4000 lx
-Shake: 3-4 times per day, and changing the positions of the flasks occasionally.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25 °C
Nominal and measured concentrations:
-Nominal concentration: 2.0, 4.0, 8.0 and 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Initial cells density: 5 x 10e5 – 6 x 10e5 cells*mL-1
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Medium used: SE medium

EFFECT PARAMETERS MEASURED
-Determination of cell concentrations: particle cell meter with microscopy
-Chlorophyll measurement: fluorescence measurement (Phyto-PAM Phytoplankton Analyzer, Walz, Germany), wavelength, 680 nm.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 (exception: 10 mg/L)

Statistic tool: SPSS 18.0 package
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
5.45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
The EC50 (96 h) was determined to be 5.45 mg/L.
Executive summary:

The effects of the test item on the growth and physiological characteristics of Chlorella pyrenoidosa were studied.

The test organism was exposed to the following test concentrations: 2.0, 4.0, 8.0 and 10.0 mg/L.

Although the test concentrations were not analytically verified, the exposure concentrations seemed to be stable during the test as both effects based on inhibition of the growth rate and the level of chlorophyll tend to increase with increasing concentration levels and exposure time.

The results revealed that the test item had acute inhibitory effects on the growth of Chlorella pyrenoidosa.

The EC50 at 96 h was determined to be 5.45 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20.07.2012-08.04.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without significant deviations
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (with and without algae) after 24 hours, 48 hours and at the end of the test. At the same sampling times, duplicate samples were also taken from the control.
Determination of algal biomass:
A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by fluorescence measurement (excitation 440 nm, emission 680 nm) (BIO-TEK(R) Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from all test concentrations to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.
Vehicle:
no
Details on test solutions:
The selection of the test concentrations was based on the results of the following second range-finding test:
Biological and analytical results of the range-finding test (0-72 hours):

Nominal Concentration [mg test item/L] Inhibition of algal biomass after 72 hours [%] Measured concentration of the test item [mg/L]
0 hours 72 hours
Negative Control 0 n.a. n.a.
0.10 12.6 0.0833 1 13.0 0.835 10 47.7 8.75 100 99.4 88.7 36.7
n.a.: not analyzed, LOQ =0.040 mg/L

The following nominal concentrations of the test item were tested: 2.2, 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item). Nominal concentrations of the test item exceeding 100 mg/L were not tested in accordance with the test guidelines.
The test design included three replicates per test concentration and six replicates of the control. Six additional replicates were prepared per treatment for analytical measurements. Three replicates were inoculated with algal cells and the other three were incubated without algae in the dark.
The test was started using a nominal algal cell density of 5000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter(R), Model Z2).
A static test design was applied. The duration of the test was 72 hours.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
The test flasks were incubated in a temperature-controlled water bath at a temperature of 21 °C.
pH:
The pH was 7.6 - 7.8 during the test period.
Dissolved oxygen:
not reported
Salinity:
Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the following concentrations:
Composition of dilution water (AAP Medium):
Ingredients Concentration
Macro-nutrients NaHCO3 250.0 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L
Trace elements H3BO3 186.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.27 µg/L
CoCl2 × 6 H2O 1.43 µg/L
CuCl2 × 2 H2O 0.012 µg/L
Na2MoO4 × 2 H2O 7.26 µg/L
FeCl3 × 6 H2O 160.0 µg/L
Na2EDTA × 2 H2O 300.0 µg/L

The test was performed in a closed system, because the test item was determined to be volatile. To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to the test water. Furthermore, the NaHCO3 concentration was increased in order to provide an additional carbon source. The modified medium was prepared according to the test guidelines and according to ISO Guidance Document [ISO, 1999]. The pH was 7.5.
Nominal and measured concentrations:
Nominal concentrations: 2.2, 4.6, 10, 22, 46 and 100 mg/L
The measured concentrations of Methyl cinnamate in the test media of the nominal test concentrations of 2.2 to 100 mg/L were between 82 and 92% of the nominal values at the start of the test. Thus, the correct dosing of the test item Methyl cinnamate was confirmed.
During the test period of 72 hours, a decrease of test item concentration in the test media occurred. In the old test samples the measured concentrations of Methyl cinnamate in the test media ranged from 47 to 63% (24 hours), from 46 to 59% (48 hours), and from 16 to 57% (72 hours, see analytical results and Table 5 in Appendix 1).
The losses during the test were considered to be due to adsorption of the test item onto the algae as well as onto the test vessels and degradation. As the test was performed in a closed system losses via volatilisation are expected to be negligible. It was observed that photolytic isomerisation took place during the test and changed the isomeric ratio of the test item from the E-Isomer more to the Z-Isomer of the test substance. These assumptions are based on results of an additional stability trial performed in parallel to the biological test where the test substance was incubated without algae in the dark and only E-methyl cinnamate and no Z-methyl cinnamate was found during the 72h testing period. Therefore, the mean measured concentration of the test item is based on the sum of the two isomers (E-methyl cinnamate and Z-methyl cinnamate).
The biological results were related to the mean measured test item concentrations (sum of the two isomers) calculated as the geometric means of the concentrations measured at the start, after 24, 48 hours and at the end of the test.
Details on test conditions:
The test flasks inoculated with algae were incubated in a temperature-controlled water bath at a temperature of 21 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 5300 Lux (range: 4710 to 5910 Lux, measured at nine places in the experimental area). The light intensity was within a ±15%-deviation from the average light intensity as recommended by the guideline.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 6.6 - 8.6
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 3.0-4.8
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The test item had a significant inhibitory effect on the growth rate µ of the algae after the test periods of 48 and 72 hours at the mean measured concentration of 4.6 mg/L and at all higher test concentrations (results of Williams t-test, one-sided smaller, α = 0.05, Table 2). The yield Y of the algae was also statistically significantly reduced at the mean measured concentration the test periods of 48 h of 11.6 mg/L and of 72 h of 4.6 mg/L (Table 3). The 48 and 72-hour NOEC based on the growth rate µ was determined to be 2.2 mg/L, since up to and including this mean measured concentration the growth rate of the algae after 48 and 72 hours was significantly lower than in the control.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in February 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.97 mg/L (Harlan Study Number D50552), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.71 - 1.7 mg/L).
Reported statistics and error estimates:
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test [Williams, "A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 1971, 27, 103-117"; Williams, "The comparison of several dose levels with a zero dose control. Biometrics 1972, 28, 519-531"] or Welch t-test [Sachs, "Applied Statistics, Springer Series in Statistics, Springer Verlag 1984, ISBN 0387909761"], where appropriate.

In the control, the biomass increased by a factor of 149 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 11%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.

The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.3%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

The pH in the control was 7.8 at test start and end fulfilling the requirement of the OECD guideline, the pH of the control medium should not increase by more than 1.5 units during the test. The pH of the test media was in the range of 7.6 to 7.8 during the test period. The water temperature during the test was maintained at 21 °C.

Validity criteria fulfilled:
yes
Conclusions:
ErC50 (72h, algae): 7.6 mg/L, ErC10 (72h, algae): 4.0 mg/L
Executive summary:

The biological results can be summarized as follows (based on geometric mean measured concentrations of the sum of the test item Z- and E-methyl cinnamate):

 

Parameter

Growth rate

Yield

(0-72 h)

EC10  (mg/L)

4.0

2.2

EC20  (mg/L)

5.0

2.8

EC50  (mg/L)

7.6

4.4

NOEC (mg/L)

2.1

2.1

LOEC (mg/L)

4.6

4.6

In conclusion, the test item methyl cinnamate had a significant inhibitory effect on the growth rate µ of the algae after the test periods of 48 and 72 hours at the mean measured concentration of 4.6 mg/L and at all higher test concentrations (results of Williams t-test, one-sided smaller, α = 0.05). The yield Y of the algae was also statistically significantly reduced at the mean measured concentration the test periods of 48 h of 11.6 mg/L and of 72 h of 4.6 mg/L.

In the control, the biomass increased by a factor of 86 over 72 hours. The validity criterion of increase of biomass by at least a factor of all validity criteria were fulfilled.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Please consult section 13 for the read-across justification.
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.26 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Corrected for molecular weight.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.35 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Corrected for molecular weight.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Corrected for molecular weight.
Conclusions:
Read-across was done from the source substane methyl cinnamate. Based on the result, and on the structural, chemical and toxicological similarities between the source and target substance, the 72h ErC50 for algae is calculated to be 8.26 mg/L and the ErC10 is 5.00 mg/L.
Executive summary:

Read-across was done from the source substane methyl cinnamate. On this source substance a study with Pseudokirchneriella subcapitata was performed in accordance with OECD guideline 201 and EU guideline C.3. In this study, significant inhibitory effect on the growth rate and yield of the algae after 48 and 72h was observed, yielding a 72h ErC50 of 7.6 mg/L and a 72h ErC10 of 4.0 mg/L.

Based on these results, and on the structural, chemical and toxicological similarities between the source and target substance, the 72h ErC50 for algae in the target substance ethyl cinnamate is calculated to be 8.26 mg/L and the ErC10 is 5.00 mg/L, taking into account the difference in molecular weight between source and target substance.

Description of key information

The EC50 (96 h) was determined to be 5.45 mg/L.

Additionally, an EC50 (96h) of 7.6 mg/L is available for the read-across substance methyl cinnamate.

Key value for chemical safety assessment

EC50 for freshwater algae:
5.45 mg/L

Additional information

A publication is available that describes a study of the effects of the test item on the growth and physiological characteristics of Chlorella pyrenoidosa. The test organism was exposed to the following test concentrations: 2.0, 4.0, 8.0 and 10.0 mg/L. Although the test concentrations were not analytically verified, the exposure concentrations seemed to be stable during the test as both effects based on inhibition of the growth rate and the level of chlorophyll tend to increase with increasing concentration levels and exposure time.

The results revealed that the test item had acute inhibitory effects on the growth of Chlorella pyrenoidosa.

The EC50 at 96 h was determined to be 5.45 mg/L.

To support the results described above a read-across was done from the source substane methyl cinnamate. On this source substance a study with Pseudokirchneriella subcapitata was performed in accordance with OECD guideline 201 and EU guideline C.3. In this study, significant inhibitory effect on the growth rate and yield of the algae after 48 and 72h was observed, yielding a 72h ErC50 of 7.6 mg/L and a 72h ErC10 of 4.0 mg/L. Based on these results, and on the structural, chemical and toxicological similarities between the source and target substance, the 72h ErC50 for algae in the target substance ethyl cinnamate is calculated to be 8.26 mg/L and the ErC10 is 5.00 mg/L, taking into account the difference in molecular weight between source and target substance.