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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:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
The endpoint in this test was the inhibition of cell multiplication.
GLP compliance:
not specified
Analytical monitoring:
no
Test organisms (species):
Microcystis aeruginosa
Details on test organisms:
TEST ORGANISM
- Common name: Blue-green algae
Test type:
static
Water media type:
freshwater
Total exposure duration:
8 d
Test temperature:
27 degree C.
pH:
7
Nominal and measured concentrations:
no details available
Details on test conditions:
TEST SYSTEM
- Initial cells density: determined turditimetrically

OTHER TEST CONDITIONS
- Adjustment of pH: test solutions were neutralized
- Light intensity and quality: On a white surface protected against daylight and exposed to constant lighting by luminescent tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] - turbidity
Duration:
8 d
Dose descriptor:
other: TT
Effect conc.:
520 mg/L
Basis for effect:
biomass
Executive summary:

Bringmann and Kuhn (1978) reported a 8 -day toxicity threshold (TT) of 520 mg/L for acetonitrile in Blue-green algae, (Microcystis aeruginosa).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. However the validity criteria for control growth was not met during the definitive study.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The control cultures in the definitive test failed to meet the 16-fold increase in biomass required, despite the method achieving the required growth rate in the previous range-finder and growth trial. Cells in the controls were also found to be elongated
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
At the beginning of the definitive bioassay, duplicate vessels were prepared for analytical sampling at 0 and 72 hours. Samples were analysed at 0 and 72 hours. Those samples for 72 hour analysis were stored in the test area under the same conditions as the test vessels prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances): The appropriate volume of acetonitrile was removed directly from its storage bottle using a micro syringe and injected into each of the test vessels. Immediately after injection of acetonitrile each test vessel was sealed leaving an approximately 11% headspace.
Test organisms (species):
other: Synechococcus leopoliensis
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 1405/1
- Source (laboratory, culture collection): The cultures was originally obtained from the Culture Collection of Algae and Protozoa (CCAP).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
22 (+/-) 2 degrees. Temperature was continuously monitored in a surrogate vessel using IceSpy (Comark) electronic environmental equipment.
pH:
0 hours - 7.01 - 7.11
72 hours - 7.10 - 8.49
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations: 100, 400, 1600, 6400 and 25600 mg/L

For discussion of the analytical results see below.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test vessels were crimp top serum vials, in the definitive test vessels (33 mL media : 4 mL headspace).
- Initial cells density: 1 x 104 cells/mL
- Replicates:
First range-finder - Two sets of three replicate test vessels (35 mL vials), each containing one 5 mm glass bead
Second rangefinder - Two sets of three replicate test vessels (150 mL), each containing one 5 mm glass bead
Growth test - Single test vessels (150 mL)
- Other: Sufficient agitation was ensured by the action of an orbital shaker set to approximately 150 rpm.

OTHER TEST CONDITIONS
- Light intensity and quality: 4440 - 8880 lux. Light intensities were recorded at 0, 24, 48 and 72 hours and test vessels were randomised daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Growth rate, Algal growth was expressed in terms of average specific growth rate and yield calculated from cell densities. Endpoints were then calculated based on nominal and mean measured concentrations.
- Determination of cell concentrations: Haemocytometer (Improved Neubauer Type)

To support the microscopic cell counts, absorbance measurements were also conducted at a wavelength of 540 nm. Phycoerytherin is a distinct pigment in Synechococcus sp. and is excited at a wavelength of 540 nm, therefore absorbance measurements may provide evidence of the presence of Synechococcus cells. Un-inoculated media at each concentration were used as the zero reference samples and the mean of two absorbance readings from pooled samples are reported.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal treatment levels 50, 500 and 5000 mg/L for rangefinder 1, 1, 10 and 100 mg/L for rangefinder 2 and 100 mg/L for the growth trial.
Reference substance (positive control):
no
Details on results:
Normal cells were seen in the control and 7 mg/L treatments after 48 hours exposure. Increasing densities of small pale cells were seen in treated levels of 28 – 1792 mg/L after 48 hours exposure. Elongated cells were observed in the control and 7 mg/L treatments after 72 hours exposure. Increasing numbers of small pale cells were present in treatments of 28 – 1792 mg/L after 72 hours. It can be observed in these examples that the stimulatory effect was very difficult to accurately quantify.

It was also noted that control cells appeared elongated by 72 hours indicating some form of stress in the test system
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The data from the definitive bioassay failed to meet the validity criteria and was therefore not subject to statistical analysis.

As a result of the consistently poor growth experienced under the sealed test conditions required for this volatile test compound, it was decided, after consultation with the Sponsor’s Representative, that the test species should be changed to a single celled species less likely to form aggregates. Synechococcus leopoliensis was chosen as a substitute cyanobacterium due to being single celled and its recommendation in the OECD guidelines.

The results are absed on the studies with Synechococcus leopoliensis.

Range-finding bioassay - 1

Cell counts were performed at 72 and 96 hours. 72 hour cell counts indicated that a 16 fold increase in biomass had not been achieved. The test was extended to 96 hours in an attempt to achieve the required increase in biomass. After 96 hours this increase had been achieved, however, exponential growth had not been sustained for 96 hours, as demonstrated by the pronounced decrease in average specific growth rate. Control cells also showed signs of elongation indicating stress and/or nutrient limitation in the test system.

Range-finding bioassay - 2

A further range-finding bioassay was performed using a method including a headspace in an attempt to improve growth rate and assess the effects of acetonitrile at lower concentrations. The control growth in this study was significantly improved and met all validity criteria by 72 hours. No significant effects were observed at 1 or 10 mg/L. The 100 mg/L treatment indicated effects thought to be either bacterial contamination or an effect causing reduction in size and pigmentation of the algal cells. Due to the small size and reduced pigment of these cells they could not be microscopically confirmed as Synechococcus leopoliensis and as such were not included in cell counts. Due to this effect a growth trial was performed to assess whether bacterial contamination was the cause of the effect observed at 100 mg/L.

Growth trial

This trial indicated that the large increase in small pale cells observed at 100 mg/L were due to affected algal cells and not bacterial contamination. Some cells were observed in un-inoculated media at treatments of 100 mg/L, however these cells were not equivalent in size, shape or frequency to the cells observed in the inoculated 100 mg/L treatment. An assumption was made after this trial that the small cells observed at treated level were due to growth stimulation and abnormal cell growth of Synechococcus leopoliensis. Therefore in this and subsequent bioassays these cells were included in the cell counts. The results of this trial indicate a stimulation of growth was observed at 100 mg/L.

Defnitive study

The definitive bioassay was performed in an attempt to establish a NOEC. Due to the difficulties associated with accurately quantifying the numbers of cells in stimulated treatments the test methods were not considered sufficiently accurate to establish ECx values. This test was performed using smaller test vessels (35 mL) than the second range-finding bioassay and growth trial (150 mL). This was due to the quantity of vessels required and space limitation in the test area. However, the same media and ratio of headspace to media volume was used to give equivalent growth conditions as those in the larger test vessels.

Analytical results - definitive study

Samples taken at the start of the definitive bioassay confirmed that acetonitrile concentrations were achieved between 76 and 104% of nominal. After 72 hours samples from replicate test vessels indicated that acetonitrile could only be maintained satisfactorily (80-120% of nominal) at concentrations of 1792 mg/L (101%). For samples at a nominal concentration of 448 mg/L an approximate 40% loss in concentration was measured. Samples at nominal concentrations of 7 – 112 mg/L indicated a measured loss close to 100%. However, vessels containing no-algal inoculum, at 112 mg/L indicated a lower rate of loss (33%) when compared to the inoculated vessels at same concentration (approximately 98% loss). The losses observed in the no-algal treatments at 7 and 1792 mg/L were comparable to those from the inoculated samples. The difference observed at 112 mg/L with and without algal inoculum may indicate that some uptake or adsorption may be taking place at lower treatment concentrations.

Samples analysed from the definitive bioassay indicate that, when using a sealed test system with headspace, exposure of acetonitrile could not satisfactorily be maintained at low treatment levels.

The following results are expressed in terms of nominal concentration of acetonitrile.

Definitive study results

Nominal conc. (mg/L)

Mean 72 h cell density (x 104cells/mL)

Mean (0-72h) average specific growth rate (day-1)

% inhibition

Co-efficient of variation

Mean absorbance at 540 nm*

Control

148.6

0.90

na

4.88

0.023

7

269.1

1.10

-22.3

1.74

0.039

28

295.2

1.13

-25.7

1.52

0.066

112

870.4

1.49

-65.6

3.70

0.114

448

829.8

1.47

-64.1

0.68

0.114

1792

725.83

1.43

-59.1

2.88

0.107

* - mean of two samples taken from each replicate test vessel at 72 hours

The results demonstrate that the control growth was limited as the 16 fold increase in biomass (160 x 104 cells/mL) was not achieved. It was also noted that control cells appeared elongated by 72 hours indicating some form of stress in the test system. The control cultures also did not maintain an exponential phase of growth for the test period showing a decline in growth rate each day. Results from all treatments showed biologically significant stimulation of growth when compared to the control cultures. The cell counts were extremely difficult to quantify, due to the stimulation of small pale cells, in treatments at and above 28 mg/L.

The absorbance readings at 540 nm (wavelength specific to the pigment distinct to this species), correlate with the observed increases in cell numbers. Whilst difficult to quantify, this is regarded as evidence that the many small cells observed were cells of Synechococcus.

The overall (0-72 hour) coefficient of variation (CoV) for the control cultures was 4.88% (< 7%) and the daily CoV was less than 35% over each period of the definitive. The average specific growth rate in the control cultures over the test period was below the 0.92 day-1 and the cultures did not therefore achieve a 16 fold increase in biomass over the test period. The control vessels also showed no evidence of achieving un-limited exponential growth.

The test was therefore considered to have failed and it is suggested that a sealed test system may not be suitable for providing adequate growth conditions with Synechococcus leopoliensis.

Validity criteria fulfilled:
yes
Remarks:
The control cultures met all required validity criteria.
Executive summary:

This study was designed to assess the effects of acetonitrile on species of cyanobacteria. Two species, Anabaena sp. and Synechococcus leopoliensis, were tested through various trials and bioassays in an attempt to accurately provide an assessment of the effects of acetonitrile.

Anabaena sp. was found to grow sub-optimally (forming aggregations) using a sealed system with no headspace. However, chemical analysis demonstrated that exposure concentrations of this volatile test item could be accurately maintained using this test system.

As a result the test species was changed to a single-celled species, Synechococcus leopoliensis, which was thought to have less probability of forming aggregates. Further trials were performed in an attempt to meet the validity criteria for control growth rate with this species.

A definitive test was initiated using the method shown to give acceptable growth in the range-finder and trial. The definitive test employed smaller vessels (due to space limitation) but with an equal ratio of headspace to media volume (33 mL media : 4 mL headspace).

Algal cultures with an initial cell density of 10 x 104 cells/mL were exposed to nominal concentrations of 7, 28, 112, 448 and 1792 mg/L. In response to the results of range finding bioassays the definitive test range was extended to a geometric series separated by a factor of 4 in an attempt to achieve the required endpoints. Test and control inocula were prepared by the addition of a defined quantity of algal stock culture to AAP growth media enriched with 100 mg/L NaHCO3 to provide a CO2 source. These inocula were then used to fill each individual test vessel. Acetonitrile was added via direct injection to each vessel and the vessel sealed, leaving an approximately 11% headspace.

The control cultures in the definitive test failed to meet the 16-fold increase in biomass required, despite the method achieving the required growth rate in the previous range-finder and growth trial. Cells in the controls were also found to be elongated, indicating possible stress in the test system. Cultures at a treated level of 7 mg/L achieved exponential cell growth and exceeded the 16-fold increase in biomass. Levels of 28 mg/L and above indicated pronounced stimulation of growth, however due to effects of reduced cell size and pigmentation reduction this stimulation was very difficult to quantify using microscopic methods.

Although difficult to quantify, under these test conditions, acetonitrile appears to provide an energy source or nutrient to provide an increase in growth rate when compared to control cultures. It is unclear as to whether this effect is a true effect of acetonitrile or an artefact produced from the insufficient growth conditions provided by the test system.

The stimulatory response observed in this study in groups treated with acetonitrile, may provide inaccurate estimations of effects. It is difficult to confidently assume that acetonitrile truly stimulates growth, or whether it provides an alternative energy source which is lacking in the growth media.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 April 2010 to 20 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Version / remarks:
2006
Deviations:
yes
Remarks:
The light intensity across the test area on days 0 and 3 exceeded the ±15% variability specified in the Test guideline. This deviation did not affect the validity or integrity of the study as all the validity criteria were met.
Principles of method if other than guideline:
The test was conducted using a static sealed test system, without headspace to avoid loss of the test substance by volatilisation from the test system.
GLP compliance:
yes
Specific details on test material used for the study:
Lot No.: SZBA021A
Purity: 100.0%
Expiry Date: 11 Jan 2012
Storage: Ambient
Analytical monitoring:
yes
Details on sampling:
Samples taken for analysis at 0 hours were removed from the test area at study initiation and stored refrigerated. Those samples for 72 hour sampling were stored in the test area under the same conditions as the test vessels. At 72 hours samples in storage and from the test area (0 and 72 hour samples respectively) were taken for analysis, duplicate samples of each were stored in the fridge in case of further analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
The appropriate volume of acetonitrile was removed directly from its storage bottle using a micro syringe and injected into each of the test vessels. Immediately after injection of acetonitrile each test vessel was sealed so that no headspace remained in the vessel.
Test organisms (species):
Phaeodactylum tricornutum
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 1052/1A
- Source (laboratory, culture collection): Brixham Environmental Laboratory (Devon, UK). These cultures were originally obtained from the Culture Collection of Algae and Protozoa (CCAP).
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
20 (+/-) 2 degrees. Temperature was continuously monitored in a surrogate vessel using IceSpy (Comark) electronic environmental equipment.
pH:
7.80 - 8.09
Dissolved oxygen:
No data
Salinity:
ISO Marine media.
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal test concentrations: 100, 400, 1600, 6400 and 25600 mg/L
Overall mean measured concentrations: 94.1, 387, 1647, 5920 and 25313 mg/L. As the measured concentrations were within 80-120% of nominal, endpoints have been reported based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilized 30 mL crimp top septum vials containing 1.5 g of 2 mm glass beads filled to the top (approximately 37 mL) and sealed so that no headspace remained were then used as the test vessels.
- Initial cells density: 1 x 104 cells/mL
- Replicates: For the range-finding bioassay, two sets of 3 replicate vessels were prepared for the control and each test concentration. For the definitive bioassay, six sets of 3 replicates for the control and three sets of 3 for each test concentration, were prepared. Test vessels were labelled with the replicate number and sequential letters; e.g. 1a, 1b, 1c and 2a, 2b, 2c were all the replicate vessels prepared for control vessels 1 and 2. Replicates a, b and c were then sacrificed for counting at 24, 48 and 72 hours respectively.
- Other: Sufficient agitation was ensured by the action of an orbital shaker set to approximately 150 rpm.

OTHER TEST CONDITIONS
- Light intensity and quality: 6000 - 10000 lux. Light intensities were recorded at 0, 24, 48 and 72 hours and test vessels were randomised daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Growth rate, Algal growth was expressed in terms of average specific growth rate and yield calculated from cell densities. Endpoints were then calculated based on nominal and mean measured concentrations.
- Determination of cell concentrations: Haemocytometer (Improved Neubauer Type)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal treatment levels 50, 500 and 5000 mg/L.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
400 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
400 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3 560 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 1604 – 7017 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9 696 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 7844 – 17597 mg/L
Details on results:
No abnormalities were observed under microscopic inspection during the test. No stimulation of growth was observed in any of the treatment groups when compared to the control cultures.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Statistical analysis was performed (using ToxCalc, 1999) on both average specific growth rates and yield after 72 hours (Appendices 4, 5, 6 and 7). A one-tailed Bonferroni t-test (p<0.05) was used to compare each treated group to the control group to obtain a NOEC and LOEC value. Point estimates (ErCx and EyCx) were then obtained using a Maximum Likelihood-Weibull analysis.

The raw data (cell counts) were compiled and average specific growth rates and yield data were calculated from the cell densities. Percentage inhibition values were also calculated from average specific growth rate and yield values.

The temperature in the test and control media at the start of the study was measured as 22.5ºC this was in excess of the 20 +/- 2ºC specified in the Study Plan. The daily mean light intensity remained within the range of 6000-10000 lux. However, the light intensity across test area on days 0 and 3, exceeded the +/- 15% variability specified in the Study Plan and Test guideline (Table 5). These Study Plan and Test Guideline deviations did not have an effect on the validity or integrity of the study, as all the validity criteria were met.

The following results are expressed for average specific growth rate and yield over the 72 hour test period in terms of nominal concentration of acetonitrile.

0-72 hour Average Specific Growth Rate

95% Confidence Limits

 

0-72 hour Yield

95% Confidence Limits

(Nominal mg/L)

(Nominal mg/L)

(Nominal mg/L)

(Nominal mg/L)

ErC10

3392

1047 – 4812

EyC10

323

7.39 – 922

ErC20

5154

2763 – 6689

EyC20

841

72.9 – 1789

ErC50

9696

7844 – 17597

EyC50

3560

1604 – 7017

LOEC

1600

N/A

LOEC

1600

N/A

NOEC

400

N/A

NOEC

400

N/A

N/A: Not Applicable

The control cultures met all required validity criteria as outlined in the Study Plan and test guidelines.

A statistically significant reduction in the growth rate and yield of algal cells was observed at the lowest test concentration (nominally 100 mg/L). However this was not considered to be of biological significance as the effect was not observed at an equal or greater level of significance in the tested concentration directly above (nominally 400 mg/L). Therefore the LOEC level has been assigned to the first statistically significant concentration where all subsequent concentrations show an equal or greater level of inhibition (nominally 1600 mg/L).

Validity criteria fulfilled:
yes
Remarks:
The control cultures met all required validity criteria.
Conclusions:
The 72 hr ErC50 and NOEC (growth rate) were calculated to be 9696 mg/L and 400 mg/L, respectively.
Executive summary:

In a Guideline GLP study (CEMR, 2010) the effect of acetonitrile on the unicellular marine algae Phaeodactylum tricornutum was assessed over 72 hours. using a static, sealed test system with no headspace. The test was conducted using a static sealed test system, without headspace to avoid loss of the test substance by volatilisation from the test system.

Algal cultures with an initial cell density of 1 x 104 cells/mL were exposed to nominal concentrations of 100, 400, 1600, 6400 and 25600 mg/L. Test and control inocula were prepared by the addition of a defined quantity of algal stock culture tomarine growth media. These inocula were then used to fill each individual test vessels. Acetonitrile was added via direct injection to each vessel and the vessel sealed, leaving no headspace.

 

Determination of the acetonitrile concentration was carried out using samples taken at 0 and 72 hours. Concentrations of acetonitrile were achieved and maintained between 90 and 108% of nominal throughout the test; giving overall mean measured concentrations of 94.1, 387, 1647, 5920 and 25313 mg/L.As the measured concentrations were within 80-120% of nominal, endpoints have been reported based on nominal concentrations.

 

Test cultures were incubated for 72 hours under constant illumination with mean light intensities ranging from 7004 - 8054 lux; temperature in the test area ranged from 18.55 – 20.21 ºC.

 

Cell densities were determined at 24, 48 and 72 hours to monitor growth throughout the test period. Results were expressed as average specific growth rate and yield over the 72 hour test period in terms of nominal concentration of acetonitrile.

After 72 hours the following effect concentrations were calculated:

ErC50(growth rate)      9696 (95% CI: 7484 - 17597) mg/L

EyC50(yield)                 3560 (95% CI: 1604 - 7017) mg/L

The "no observed effect concentration" (NOEC) and "lowest observed effect concentration" (LOEC) for both growth rate and yield where statistically derived as 400 and 1600 mg/L respectively.

The use of a sealed test system effectively maintained the concentrations of the volatile test item over the duration of this study, as indicated by measured concentration of 90-108% of nominal. As a result this test is regarded as an indication of the worst case scenario with regards to the effect of acetonitrile on this algal species.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
The endpoint in this test was the inhibition of cell multiplication.
GLP compliance:
not specified
Analytical monitoring:
no
Test organisms (species):
Scenedesmus quadricauda
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
Test type:
static
Water media type:
freshwater
Total exposure duration:
8 d
Test temperature:
27 degree C.
pH:
7
Nominal and measured concentrations:
No details available
Details on test conditions:
TEST SYSTEM
- Initial cells density: determined turditimetrically

OTHER TEST CONDITIONS
- Adjustment of pH: test solutions were neurtralized
- Light intensity and quality: On a white surface protected against daylight and exposed to constant lighting by luminescent tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] - turbidity
Duration:
8 d
Dose descriptor:
other: TT
Effect conc.:
7 300 mg/L
Basis for effect:
biomass
Executive summary:

Bringmann and Kuhn (1978) reported an 8 -day toxicity threshold (TT) of 7300 mg/L for acetonitrile in Green algae, (Scenedesmus quadricauda).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
A closed system algal toxicity test technique with no headspace to avoid loss of volatile test substances.
GLP compliance:
not specified
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Dissolved oxygen:
Beginning of the test approximately I to 2 mg/L.
Nominal and measured concentrations:
The measured concentration is not a practical representation for the amount of toxicant applied in the test because vacuum filtration may cause considerable losses of volatile material. Hence the toxicant concentrations presented in this work are in the form of nominal concentration. Concentration controls have been conducted periodically without the addition of algal inoculum and analyzed by the total organic carbon analyzer. The difference between the nominal and measured concentrations was found to be less than 8% with a normal range of 3 to 5%.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300-ml BOD test bottles.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: No head space
- Orbital shaker: operated at 100 rpm.
- Initial cells density: 15,000 cells/ml

GROWTH MEDIUM FOR INCUBATION
- Standard medium used for: yes, the growth medium composition is the same as that described by the U.S. Environmental Protection Agency (U.S. EPA) bottle technique. NaNO3, K2HPO3, and ethylenediaminetetraacetic acid contents were reduced to 12.75 mg/L, 0.52 mg/L, and 30 ug/L, respectively.
- supplied continuously by a variable-speed pump. Air agitation was used to achieve adequate mixing.
- 4-L transparent chemostat incubator.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was stripped by nitrogen gas to reduce the dissolved oxygen (DO)level

OTHER TEST CONDITIONS
- Temperature: 24 (+/-) 1 degree C
- Light intensity and quality: 65 uEmˉ²sˉ ¹, (+/-10%).
Reference substance (positive control):
yes
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
7 943 mg/L
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5 926 mg/L
Basis for effect:
other: Dissolved Oxygen production
Details on results:
- Exponential growth in the control (for algal test): yes
Executive summary:

Chen et al (2005) reported 48 -hour, static EC50 values of 5926 mg/L (based on dissolved oxygen production) and 7943 mg/L (based on growth rate) for acetonitrile in Green algae, (Raphidocelis subcapitata, formerly known as Selenastrum capricornutum). The methods were designed to eliminate head space, and thus avoid loss of test substance by volatilization. This method is reported to be more sensitive than the conventional (open) algal batch tests.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 October 1995 - 29 March 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Physical state: colourless and transparent liquid
- Analytical purity: 100.0% (GC)
- Lot/batch No.: ESH3487
- Stability under test conditions: Stable
- Storage condition of test material: In the refrigerator
Analytical monitoring:
yes
Details on sampling:
- Sampling: 0, 72h
Vehicle:
not specified
Details on test solutions:
A set of test solutions used in this study were prepared by diluting a stock solution (100000 mg/L), with the OECD medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIES-35
- Method of cultivation: This strain was repeatedly-subcultured under sterile condition.


ACCLIMATION
- Acclimation period: 3days before starting test
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed: None
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.8 - 23.2℃
pH:
7.7 - 8.0
Nominal and measured concentrations:
nominal: 0, 1000 mg/L
measured: (0 h) <7, 996 mg/L
(72 h) <7, 499 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: breathable silicon cap
- Material: glass
- size: 300mL
- fill volume: 100 mL
- Initial cells density: 1X10^4 cells/mL
- Control end cells density: 3.6X10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes (OECD medium)


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter Counter)


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 500, 1000, 2000 mg/L
- Results used to determine the conditions for the definitive study: 72h; 0 mg/L: 100%, 500 mg/L: 121%, 1000 mg/L: 109%, 2000 mg/L: 104%
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
72h EC50 (biomass) >1000 mg/L (Nominal concentration)
24-48h EC50 (growth rate) >1000 mg/L (Nominal concentration)
24-72h EC50 (growth rate) >1000 mg/L (Nominal concentration)
72h NOEC (biomass) >1000 mg/L (Nominal concentration)
24-48h NOEC (growth rate) >1000 mg/L (Nominal concentration)
24-72h NOEC (growth rate) >1000 mg/L (Nominal concentration)
Results with reference substance (positive control):
- 72h EbC50: 0.39 mg/L

Table 1. Cell Density of Selenastrum capricornutum

Nominal concentration
(mg/L)
No.

Cell density (cells/mL)

0 hour 24 hours 48 hours 72 hours
Control 1 10,000 98,000 948,400 3,525,000
2 10,000 110,200 1,004,000 3,798,200
3 10,000 127,600 1,027,400 3,609,400
Average 10,000 111,933 993,267 3,644,200
S.D. 0 14,876 40,579 139,885
1000 1 10,000 150,400 943,600 3,711,800
2 10,000 139,400 947,800 3,443,400
3 10,000 164,600 1,042,200 3,812,600
Average 10,000 151,467 977,867 3,655,933
S.D. 0 12,634 55,754 190,835

             

                           

Table 2. Growth Inhibition of Selenastrum capricornutum

Concentration Area Inhibition(%) Rate Inhibition(%) Rate Inhibition(%)
mg/L No. A (0-72h) IA (0-72h) μ (24-48h) Im (24-48h) μ (24 -72h) Im (24-72h)
Control 1 66814000 - 0.0946 - 0.0746 -
2 71719000 - 0.0921 - 0.0737 -
3 70433000 - 0.0869 - 0.0696 -
Average 69655000 - 0.0912 - 0.0726 -
1000 1 70198000 - 0.0765 - 0.0668 -
2 66814000 0.0799 0.0668
3 74114000 0.0769 0.0655
Average 70375000 -1.0 0.0778 **14.7 0.0664 *8.5

*: 5%, **: 1% Significant level       

      

      

Conclusions:
The 72 hr EC50 and NOEC (growth rate) were both concluded to be >1000 mg/L.
Executive summary:

In a guideline (OECD 201) and GLP study, MCSI of Japan (1996) reported the following NOEC values for acetonitrile in alga (Selenastrum capricornutum):

72 -hr NOEC (biomass): >1000 mg/L

24 -48 hr NOEC (growth rate): >1000 mg/L

24 -72 hr NOEC (growth rate): >1000 mg/L

Description of key information

Reported toxicity values for acetonitrile in freshwater algae range from 520 mg/L (8-day TT in the blue-green algae Mictocystis aeruginosa) to 7943 mg/L (48-hr EC50 in the green algae Raphidocelis subcapitata).  A 72-hr NOEC of 400 mg/L was reported in a Guideline study of the marine algae Phaeodactylum tricornutum.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
520 mg/L
EC10 or NOEC for marine water algae:
400 mg/L

Additional information

The toxicity of acetonitrile has been studied in freshwater and marine algae. A summary of the available data is provided in the table below. Toxicity threshold values based on the first detectable inhibition of cell multiplication for the green algae (Scenedesmus quadricauda) and for the blue-green algae (Microcystis aeruginosa) has been reported by Bringmann and Kühn as 7300 and 520 mg/L respectively.

 

Chen et al (2005) reported 48 -hour, static EC50 values of 5926 mg/L (based on dissolved oxygen production) and 7943 mg/L (based on growth rate) for acetonitrile in Green algae, (Raphidocelis subcapitata). The methods used were designed to eliminate head space, and thus avoid loss of test substance by volatilization. This method is reported by the investigators to be more sensitive than the conventional (open) algal batch tests.

In a guideline (OECD 201) GLP study, MCSI of Japan (1996) reported the 72 -hr NOEC values (biomass, growth rate) for acetonitrile in alga (Selenastrum capricornutum) to be >1000 mg/L.

In a Guideline GLP study (CEMR, 2010) the effect of acetonitrile on the unicellular marine algae (Phaeodactylum tricornutum) was assessed over 72 hours. using a static, sealed test system with no headspace to avoid loss of the test substance by volatilisation from the test system. After 72 hours the following effect concentrations were calculated:

ErC50(growth rate)      9696 (95% CI: 7484 - 17597) mg/L

EyC50(yield)                 3560 (95% CI: 1604 - 7017) mg/L

The "no observed effect concentration" (NOEC) and "lowest observed effect concentration" (LOEC) for both growth rate and yield where statistically derived as 400 and 1600 mg/L respectively.

Toxicity of acetonitrile to algae

SPECIES

TEST TYPE

Duration

TOXICITY

END POINT

(mg/l)

REFERENCE

Raphidocelis subcapitata(green algae)

Static, nominal concentration,

no head space

48 hr

EC50= 5926

Disolved oxygen production

Chen et al (2005)

Raphidocelis subcapitata(green algae)

Static, nominal concentration,

no head space

48 hr

EC50= 7943

Growth rate

Chen et al (2005)

Microcystis aeruginosa

(blue-green algae)

Static, nominal concentration

8 days

TT = 520

Bringmann and Kühn (1978)

 

Scenedesmus quadricauda

 (green algae)

Static, nominal concentration

8 days

TT = 7300

Bringmann and Kühn (1978)

 

Selenastrum capricunutum

(green algae) 

 Static, nominal concentration

 72 hr

 NOEC > 1000

 MCSI, Japan (1996)

Phaeodactylum tricornutum

(marine algae)

 Static, nominal

concentration,

no head space

 72 hr

NOEC = 400 

CEMR (2010) 

TT = toxicity threshold for inhibition of cell multiplication.