4-chlorobenzonitrile

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.06.2014 - 16.06.2014

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)

Analytical monitoring:

yes

Details on sampling:

In the main test samples were taken from all concentrations and from the control vessel at test start (day 0) and 24, 48 and 72 hours after beginning of the test. All test item concentrations were analysed at 0 and 72 hours to verify test concentrations.

Vehicle:

no

Details on test solutions:

The test was performed with the following range of test item concentrations (spaced by a geometric factor of 2.0): 6.25, 12.5, 25.0, 50.0 and 100 mg/L 4-Chlorobenzonitrile and a control. There were six replicates for the control and three replicates for each of the test item concentrations. The cell density was adjusted to an initial concentration of 0.5 × 104 cells/mL in each test vessel.
The necessary amount of test item for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was treated with ultrasonic sound for 10 minutes. After a stirring period of 24 hours the solution was treated again with ultrasonic sound for 25 minutes. The further concentrations were made by diluting the appropriate solutions to give the required test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindak
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, 37075 Göttingen, Germany
- Method of cultivation: The algae were grown semi-continuously in the laboratory in liquid cultures under permanent illumination. Old medium was periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures were ordered regularly from the culture collection. Culture conditions were as follows:
- Illumination: from the top by light tubes, 60 – 120 µE m-2 s-1 at cell culture level
- Temperature: 21 – 24 °C
- Culture flasks: 100 mL Erlenmeyer flasks
- Visually healthy cells were used for the test
- CO2 supply: by shaking on rotating shaker (approx. 105 rpm)
Cells from this semi-continuous liquid stock culture were used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observations were recorded.

Hardness:

no data

Test temperature:

21.0 - 23.7 °C

pH:

7.06 - 8.10

Dissolved oxygen:

no data

Salinity:

no data (freshwater)

Nominal and measured concentrations:

Nominal test item concentration: 0, 6.25, 12.5, 24.0 50.0, 100 mg/L
Measured test item concentrations after 0 h: 5.40, 11.1, 23.2, 45.1, 93.2 mg/L
Measured test item concentrations after 72 h: 1.16, 3.54, 9.70, 31.4, 70.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps and two baffles
- Type: closed
- Initial cells density: 0.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes (AAP-Medium, according Annex 3 of OECD 201)

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: pH of the test medium was adjusted to 7.5 +/- 0.1
- Photoperiod / Light intensity and quality: Illumination continuously from the top, 63 - 100 µEm^-2s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined by means of a calibration curve, where cell numbers (x axis) were plotted versus fluorescence signals (y axis). To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata. The daily fluorescence measurements were performed with a Tecan Reader (infinite 200Pro; serialnumber 1305001441) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.10.4.0).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

RANGE FINDING STUDY
- Test concentrations: 0, 0.01, 0.10, 1.0, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: As the dose response range was observed within a range of 10 to 100 mg/L, the main test concentrations were chosen to be 0, 6.25, 12.5, 25.0, 50.0 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

51.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

Test species Pseudokirchneriella subcapitata (intial concentrations about 0.5 x 10^4 cells/mL) were exposed in a static test system for 3 days to 5 test concentrations of Potassium dichromate (0.0512, 0.128, 0.320, 0.800 and 2.00 mg/L, serial dilution factor: 2.5). Six replicates were performed for the control and three for each test item concentration. After 1, 2 and 3 days, the cell growth was determined by flourescence detection. The mean value of the cell concentration was plotted versus time to produce growth curves for each concentration.
The EC50 value for growth rate, yield and biomass was calculated to be 1.20, 0.751 and 0.837 mg/L, respectively. The EC50 values were considered to be within an acceptable range therefore it can be considered that the algae cells are sentitive.
Dates of work: 17.02.2014 - 20.02.2014

Reported statistics and error estimates:

The statistical evaluation for 3 days was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was performed by calculating the Levene Test. The NOEC and LOEC were determined by Bonferroni-Holms corrected Welch-test (left sided). The ErC10, 20, 50 values for growth rate and yield were determined by the probit analysis following the Gompertz distribution.
Statistically significant inhibitory effects were observed after 72 hours at the test item concentration of 50.0 and 100 mg/L for growth rate and yield based on nominal test item concentrations, and 37.5 and 81.0 mg/L based actual concentrations.

The average cell numbers for each concentration and time of sampling are shown in table 1. Table 2 shows the percentage inhibiton of growth rate and table 3 the percentage inhibition of yield.

Table 1: Average cell number for each sampling time and concentration.

Test item concentration	Average cell numbers/mL^1)
[mg/L]	0d	1d	2d	3d
[LM1] 0	0.45	1.17	5.19	22.73
6.25	0.51	1.22	5.85	17.80
12.5	0.45	1.06	5.45	16.91
25.0	0.28	0.92	4.03	12.50
50.0	0.18	0.37	0.55	0.91
100	0.07	0.07	0.17	0.23

1) Algae counts are divided by 10000. At the start, the cell density was adjusted to 0.5 x 10^4 cells/mL.

Table 2: Percentage inhibition of growth rate

Test item concentration	% Inhibition of growth rate
[mg/L]	0d - 1d	0d - 2d	0d - 3d
Control	0.0	0.0	0.0
6.25	6.9	-1.7	8.6
12.5	8.6	-3.6	7.1
25.0	-28.6	-10.8	4.8
50.0	24.7	56.0	62.7
100	6.5	61.7	68.5

Table 3: Percentage inhibition of yield

Test item concentration	% Inhibition of yield
[mg/L]	1d - 0d	0d - 2d	0d - 3d
Control	0.0	0.0	0.0
6.25	1.4	-12.7	22.4
12.5	15.3	-5.5	26.1
25.0	9.7	20.9	45.2
50.0	73.6	92.2	96.8
100	100.0	97.9	99.3

The morphology of the algae cells was observed microscopically. The cells were considered normal for the control and all test item concentrations. The cell number declined by increasing test item concentrations. At 25.0 mg/L the cells were slightly pale and at the two highest test item concentrations no cells were seen anymore.

The test was performed under static conditions. The concentration of the active ingredient was verified in test medium by analysing the content of 4-Chlorobenzonitrile in the samples at test start (0 h) and at test end (72 h aged). The results are presented in Table 4.

Table 4: Concentrations of 4 -Chlorobenzonitrile in the test medium.

Test item	4-Chlorobenzonitrile	Sampling time	4-Chlorobenzonitrile measured
nominal [mg/L]	nominal [mg a.i./L]		[mg /L]	% of nominal	Geometric mean %	actual Test Item [mg/L]^1)
0	0	0 h	n.d.	-
		72 h	n.d.	-
6.25	6.23	0 h	5.40	87	41	2.56
		72 h	1.16	19
12.5	12.5	0 h	11.1	89	50	6.25
		72 h	3.54	28
25.0	24.9	0 h	23.2	93	60	15.0
		72 h	9.70	39
50.0	49.9	0 h	45.1	90	75	37.5
		72 h	31.4	63
100	100	0 h	93.2	93	81	81.0
		72 h	70.8	71

Limit of quantification (LOQ) = 1.00 mg/L 4-Chlorobenzonitrile

¹⁾Conc. based on geomean of each test item concentration

n.d.= not detectable (≙0.300 mg/L 4-Chlorobenzonitrile)

The initial mean measured concentrations of 4-Chlorobenzonitrile in the test item solutions was 87 - 93 % of nominal concentration. The measured concentration of the aged samples ranged between 19 and 71 % of nominal.

Since the initial measured concentrations of 4-Chlorobenzonitrile in the test item solutions were above 80 % and below 120 % and the concentration in the aged samples were below 80 % the biological endpoints were evaluated using the nominal and actual concentration based on the geometric mean of each test item concentration.

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects for 4-Chlorobenzonitrile were observed at 50.0 and 100 mg/L (nominal) for growth rate and yield, respectively. Therefore the overall LOEC was determined to be 50.0 mg/L (nominal) and the overall NOEC was 25.0 mg/L (nominal) for growth rate and yield. The 72h-EC50 value for growth rate (ErC50) was 51.9 mg/L (nominal). The 72h-EC50 value for yield (EyC50) was 20.5 mg/L (nominal).

Executive summary:

A study was performed in order to evaluate the toxic potential of 4 -Chlorobenzonitrile towards Pseudokirchneriella subcapitata according to OECD Guideline No. 201 ("Freshwater Alga and Cyanobacteria, Growth Inhibition Test"). Initial concentration of 0.5 × 10⁴ cells/mL in each test vessel, were exposed in a static test system for 3 days. A static test with nominal test item concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg/L 4-Chlorobenzonitrile was performed. Six replicates were used for the control and three for each test item concentration. After 1, 2 and 3 days, the cell density was determined by fluorescence detection. The mean value of the cell concentration was plotted versus time to produce growth curves for each concentration.The EC₅₀, EC₂₀andEC₁₀values as well as LOEC and NOEC values were evaluated after 72 h test period.

Significant inhibitory effects for 4-Chlorobenzonitrile were observed at 50.0 and 100 mg/L (nominal) for growth rate and yield, respectively. Therefore the overall LOEC was determined to be 50.0 mg/L (nominal) and the overall NOEC was 25.0 mg/L (nominal) for growth rate and yield. The 72h-EC50 value for growth rate (ErC50) was 51.9 mg/L (nominal). The 72h-EC50 value for yield (EyC50) was 20.5 mg/L (nominal).

Reference 2

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:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

Chlorella vulgaris

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

6 h

Duration:

6 h

Dose descriptor:

EC50

Effect conc.:

67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: photometric determination of biomass at wavelength of 680 nm

Duration:

6 h

Dose descriptor:

EC50

Effect conc.:

72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: photometric determination of biomass at wavelength of 750 nm

Validity criteria fulfilled:

not specified

Conclusions:

The 6-h EC50 (basis for effect: biomass) of 4-chlorobenzonitrile for Chlorella vulgaris was determined to be 67 mg/L (photometric determination of wavelength at 680 nm).

Executive summary:

The effect of 4-chlorobenzonitrile on reduction of biomass of Chlorella vulgaris was determined photometrically at 680 nm and 750 nm. No data are available on the concentrations tested. Chlorella vulgaris was used in aqueous solution of the test substance at approximately 7.5 x 10^8 cells/ml. The cell suspensions were incubated for 6 hours at 36.5 °C at an illumination intensity of 28 W/m².

The 6-h EC50 was determined to be 67 mg/L and 72 mg/L at 680 nm and 750 nm, respectively.

Description of key information

Significant inhibitory effects for 4-Chlorobenzonitrile were observed at 50.0 and 100 mg/L (nominal) for growth rate and yield, respectively. Therefore the overall LOEC was determined to be 50.0 mg/L (nominal) and the overall NOEC was 25.0 mg/L (nominal) for growth rate and yield. The 72h-EC50 value for growth rate (ErC50) was 51.9 mg/L (nominal). The 72h-EC50 value for yield (EyC50) was 20.5 mg/L (nominal).
In a second study the 6-h EC50 (basis for effect: biomass) of 4-chlorobenzonitrile for Chlorella vulgaris was determined to be 67 mg/L (photometric determination of wavelength at 680 nm).

Key value for chemical safety assessment

EC50 for freshwater algae:

51.9 mg/L

Additional information

A study was performed in order to evaluate the toxic potential of 4 -Chlorobenzonitrile towards Pseudokirchneriella subcapitata according to OECD Guideline No. 201 ("Freshwater Alga and Cyanobacteria, Growth Inhibition Test").

Significant inhibitory effects for 4-Chlorobenzonitrile were observed at 50.0 and 100 mg/L (nominal) for growth rate and yield, respectively. Therefore the overall LOEC was determined to be 50.0 mg/L (nominal) and the overall NOEC was 25.0 mg/L (nominal) for growth rate and yield. The 72h-EC50 value for growth rate (ErC50) was 51.9 mg/L (nominal). The 72h-EC50 value for yield (EyC50) was 20.5 mg/L (nominal).

In a second study the effect of 4-chlorobenzonitrile on reduction of biomass of Chlorella vulgaris was determined photometrically at 680 nm and 750 nm. No data are available on the concentrations tested. Chlorella vulagris was used in aqueous solution of the test substance at approximately 7.5 x 10^8 cells/ml. The cell suspensions were incubated for 6 hours at 36.5 °C at an illumination intensity of 28 W/m². The 6-h EC50 was determined to be 67 mg/L and 72 mg/L at 680 nm and 750 nm, respectively. Because there were no further details on the study performance found and the incubcation period was only 6 hours this test was not considered for classification.