Pyridinium dichromate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-07-29 to 2010-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Following nominal concentrations were measured: 0.000, 0.006, 0.016, 0.050, 0.140, 0.400, 1.200, 3.400 and 10.000 mg/L
- Sampling method: At the start and at the end of the incubation period. The concentrations of the test substance were determined in aliquots of the blank containing test substance, but no algae; of one replicate of each of the test and control cultures (after filtering through a 0.2 µm filter)
- Sample storage conditions before analysis: deep frozen

Vehicle:

no

Details on test solutions:

- Method: A first stock solution (ST1) with a nominal test substance concentration of 1111 mg/L was prepared by dissolving 111.8 mg test substance in 100 mL of nutrient medium by manual homogenisation. To obtain a second stock solution (ST2, nominal 11.1 mg/L) which was used for the test, 10 mL of ST2 were filled up to 1000 mL with nutrient medium. Aliquots of ST2 were diluted with nutrient medium to obtain lower concentrations, nominally spaced by a factor of 3.
- Controls: Blank control: For the negative control group only nutrient medium was used. Positive control: Performed with the reference substance potassium dichromate.
- Chemical name of vehicle: no vehicle was used.
- Evidence of undissolved material: No.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: ATCC (American Type Culture Collection) 22662.
- Source: LGC Promochem GmbH, Germany
- Method of cultivation: Stock cultures, initially cultivated from deep frozen algae, are continuously maintained in 250 mL conical flasks (Erlenmeyer), each containing 100 mL nutrient medium, at a temperature in the range of 21 to 24 (± 2) °C under permanent light with an intensity between 4400 and 8800 lux. In about weekly intervals 1 mL of the stock culture is diluted 100-fold with nutrient medium for precultivation and incubation is continued.

ACCLIMATION
- Acclimation period: Precultures were prepared by incubating a new stock culture for four days.
- Acclimation conditions: Same as test.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

not reported

Test temperature:

22 °C

pH:

The pH was between 7.3 and 7.9 at the start of the incubation in the test cultures and it was 7.8 in the control cultures.
After 72 hours of incubation the pH was between 7.7 and 8.3 in the test cultures and it was 8.6 in the control cultures.
The pH in the control cultures changed by 0.8 units during the 72 hours of incubation, i.e. within the maximum change of 1.5 recommended by the guideline.

Dissolved oxygen:

Not reported.

Nominal and measured concentrations:

Nominal: 0.000 (Control), 0.006, 0.016, 0.050, 0.140, 0.400, 1.200, 3.400, 10.000 mg/L
Actual concentrations at start of test: Actual concentrations at end of test: Geometric mean of actual/calculated concentrations: < QL (Control), 0.01 (calculated), 0.02 (calculated), 0.05 (calculated), 0.13 (calculated), 0.38, 0.79, 2.85, 8.48 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL closed Erlenmeyer flasks filled with 100 mL medium .
- Initial cells density: 10000 cells/mL
- Control end cells density: the cell density in the control cultures increased by a factor of about 99.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes.
- Sterile deionised water and sterile concentrated nutrient medium 9 + 1 was used.
- Conductivity of the deionised water: < 5 µS/cm


OTHER TEST CONDITIONS
- Sterile test conditions: The test cultures are prepared under steril conditions.
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 5480 - 6550 lux, maximum deviation from mean light intensity was 11 %


EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic cell counter with Casy Cell Counter after 24, 48, and 72 hours of incubation
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.6
- Justification for using less concentrations than requested by guideline: not applicable.
- Range finding study: yes .
- Test concentrations: . The test substance concentrations used in the main experiment were chosen according to the results of a preliminary range finding experiment (non GLP). In this preliminary study four test substance concentrations, nominal 100, 10, 1, and 0.1 mg/L, were tested. One negative control culture (nutrient medium only) was set up concurrently. One replicate culture for each concentration and six replicates for controls were tested. Measurements of cell densities were made after 24, 48, and 72 hours.
- Results used to determine the conditions for the definitive study: Based on the inhibition of the algal yield and the growth rates, the range finding study revealed EC50 values between <0.1 and 1.1 mg/L.

Reference substance (positive control):

yes

Remarks:

72h EC50 (K2Cr2O7): for growth rate 1.32 mg/L and yield 0.62 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.05 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.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: During the 72 hours incubation period the cell density in the control cultures increased by a factor of about 99, corresponding to about 6.6 generations.
- Observation of abnormalities: none
- Colour differences: No precipitation of test substance was noted during the incubation period. The test media containing the three highest test substance concentrations were coloured light yellow during the first 48 hours of exposure. At the end of the test light yellow colouration was only observed in the two highest test media. All other test and control media were colourless throughout the exposure period.
- Any stimulation of growth found in any treatment: yes
- Effect concentrations exceeding solubility of substance in test medium: no

Growth Inhibition:
- Based on the yield and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 100.5 % inhibition to 7.5 % enhancement.
- Based on the average growth rates and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 100.0 % inhibition to 1.5 % enhancement.

Following results were determined:
based on the yield NOEC = 0.05 mg/L
based on the average growth rates NOEC = 0.13 mg/L
based on the yield EC50 = 0.14 mg/L
based on the average growth rates EC50 = 1.05 mg/L

Results with reference substance (positive control):

The last reference test with potassium dichromate was conducted from the 21st to the 24th of June 2010 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg potassium dichromate, respectively. These results establish the reliability of the test procedures for this kind of study type.

Reported statistics and error estimates:

Based on the yield as well as on the average growth rates two "lowest observed effective concentrations" (LOECs) are calculated by comparison of the data of the three replicates of each test substance culture with the negative control (analysis of variance, followed by the Scheffé-test, P = 0.05). The "no observed effect concentrations" (NOECs) are then derived from these results (highest concentration with no statistically significant difference to the control).

Table 1: Test substance concentrations

Nominal test substance concentration   (mg/L)	Actual concentrations of the test substance						Actual/Calculated geometric mean test substance concentration*)   (mg/L)
	Start of incubation			After 72 hours of incubation
	Sample no.1 and 2 (mg/L)	Mean (mg/L)	% of nominal start conc.	Sample no.1 and 2 (mg/L)	Mean (mg/L)	% of nominal start conc.
Control	<QL <QL	-	-	<QL <QL	-		-
0.006	<QL <QL	-	-	<QL <QL	-	-	0.01**)
0.016	<QL <QL	-	-	<QL <QL	-	-	0.02**)
0.050	<QL <QL	-	-	<QL <QL	-	-	0.05**)
0.140	<QL <QL	-	-	<QL <QL	-	-	0.13**)
0.400	0.38 0.38	0.38	95.0	0.39 0.39	0.39	97.5	0.38
1.200	0.61 0.60	0.61	50.4	1.02 1.02	1.02	85.0	0.79
3.400	2.65 2.65	2.65	77.9	3.05 3.06	3.06	89.9	2.85
10.000	8.75 8.76	8.76	87.6	8.73 7.70	8.22	82.2	8.48
10.000 (blank without algae)	9.52 9.53	9.53	95.3	8.31 8.31	8.31	83.1	8.90

 

QL: Quantitation limit of the analytical method used in nutrient medium (0.31 mg/L)

*) Calculated values are derived from the next higher actual/calculated test substance concentration divided by the factor 2.9 (which is the mean actual factor between the four highest geometric mean test substance concentrations

**) Calculated values

 

Table 2: Mean yield, average specific growth rates and percent inhibition

Mean actual test substance concentration (mg/L)	Yield (cells x 10^3/mL), 0-72 h		Growth Rate (per day) 0-72 h		Percent Inhibition based on
	Mean	SD	Mean	SD	Yield	Growth Rates
0 (control)	977.7	69.8	1.53	0.02	0.0	0.0
0.01	920.2	148.2	1.51	0.05	5.9	1.4
0.02	921.0	115.1	1.51	0.04	5.8	1.4
0.05	1050.8	94.9	1.55	0.03	-7.5	-1.5
0.13	499.6	279.0	1.28	0.17	48.9	16.3
0.38	407.0	320.2	1.18	0.24	58.4	22.6
0.79	173.8	21.3	0.97	0.04	82.2	36.7
2.85	3.2	3.7	0.08	0.09	99.7	94.6
8.48	-4.5	5.7	n.a.	n.a.	100.5	100.0*)

 

n.a.: not applicable: for calculation of growth rates the logarithm of the cell density is used. This is not applicable with negative values (which indicate decrease of the initial cell count).

*) Since the calculation of the growth rates of the test culture containing the highest test substance concentration is not possible, the percent inhibition was manually set to 100 %.

 

 

Table 3: Cell densities, yield, and growth rate

Mean actual test substance concentration (mg/L)	Cell Density (cells x 10^3/mL)							Yield (cells x 10^3/mL)	Growth Rate (per day)
	1. rep.	2. rep.	3. rep.	4. rep.	5. rep.	6.rep.	Mean	Mean	Mean
	incubation time: 24 h							0-24 h	0-24 h
0 (control)	38	51	37	46	43	50	44	34	1.48
0.01	45	41	39	-	-	-	42	32	1.43
0.02	41	39	48	-	-	-	43	33	1.45
0.05	51	45	49	-	-	-	48	38	1.57
0.13	50	46	47	-	-		48	38	1.56
0.38	38	51	36	-	-	-	42	32	1.42
0.79	26	25	28	-	-	-	26	16	0.96
2.85	20	9	9	-	-	-	12	2	0.15
8.48	9	11	14	-	-	-	11	1	0.09
	incubation time: 48 h							0-48 h	24-48 h
0 (control)	207	212	185	220	196	252	212	202	1.57
0.01	214	235	228	-	-	-	226	216	1.68
0.02	196	225	214	-	-	-	212	202	1.60
0.05	206	205	215	-	-	-	209	199	1.46
0.13	199	155	146	-	-	-	166	156	1.24
0.38	148	157	159	-	-	-	155	145	1.32
0.79	72	71	78	-	-	-	74	64	1.03
2.85	9	5	4		-	-	6	-4	-0.71
8.48	1	-1	3	-	-	-	1	-9	n.a.
	incubation time: 72 h							0-72 h	48-72 h
0 (control)	976	998	954	890	1006	1102	988	978	1.54
0.01	803	895	1093	-	-	-	930	920	1.41
0.02	826	1054	912	-	-	-	931	921	1.48
0.05	1121	951	1110	*			1061	1051	1.62
0.13	831	364	334	-	-	-	510	500	1.04
0.38	220	245	786	-	-	-	417	407	0.81
0.79	203	161	188	-	-	-	184	174	0.91
2.85	17	12	10	•	-	-	13	3	0.81
8.48	8	-1	10	-	-	-	6	-4	n.a.

 

n.a.: not applicable. For calculation of growth rates the logarithm of the cell density is used. This is not applicable with negative values (which indicate decrease of the initial cell count).

 

 

Table 4: pH of the test cultures and of the controls

Mean actual test substance concentration (mg/L)	pH
	at start of incubation	after 72 hours of incubation
0 (control)	7.8	8.6
0.01	7.8	7.8
0.02	7.8	8.3
0.05	7.8	8.2
0.13	7.9	7.8
0.38	7.8	7.7
0.79	7.8	7.7
2.85	7.7	7.7
8.48	7.3	7.7

 

 

Validity criteria fulfilled:

yes

Conclusions:

The test substance was tested to Pseudokirchneriella subcapitata in a static test system according to OECD 201 (2006) under GLP conditions. Under the given conditions of this study the 72 h ErC50 value was determined to be 1.05 mg/L and the NOEC was determined to be 0.13 mg/L.

Executive summary:

The influence of the test material on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata was tested according to OECD 201 (2006) under static and GLP conditions. Eight different concentrations of the test substance between nominal 0.006 and 10000 mg per litre nutrient medium, spaced by a factor of about 3.0, were tested against one concurrent negative control (nutrient medium only). Three replicates were used for each test culture and six replicates for the control culture. The cell count of the algae was about 10000 cells/mL at the start of the exposure in each vessel. In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the beginning and at the end of the incubation period. Visual observations on the appearance of the algal cultures were made each time when the cell densities were determined. Possible test substance effects were determined by comparison of the yield and of the growth rates. At the start and at the end of the experiment, samples of the cultures exposed to the test substance, of the control cultures and also of the blank with the highest concentration of the test substance, but no algae, were taken and analysed in duplicate by HPLC. The geometric mean concentrations of the test substance were determined to be 0.01, 0.02, 0.05, 0.13, 0.38, 0.79, 2.85, 8.48 and 8.90 mg/L. The test substance concentrations have not been maintained within 80 % of the nominal concentrations throughout the duration of the test. Therefore the presentation of the growth curves and the calculation of the effective concentrations were based on the mean measured test substance concentrations. No precipitation of test substance was noted during the incubation period. The test media containing the three highest test substance concentrations were coloured light yellow during the first 48 hours of exposure. At the end of the test light yellow colouration was only observed in the two highest test media. All other test and control media were colourless throughout the exposure period. A reference test with potassium dichromate was conducted to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg/L, respectively. These results establish the reliability of the test procedures for this kind of study type. Under the given conditions of this study the 72 h ErC50 value of the test substance was determined to be 1.05 mg/L and the NOEC was determined to be 0.13 mg/L.

Description of key information

The test substance was tested to Pseudokirchneriella subcapitata in a static test system according to OECD 201 (2006) under GLP conditions. Under the given conditions of this study (reference 6.1.5-1) the 72 h ErC50 value was determined to be 1.05 mg/L and the NOEC was determined to be 0.13 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.05 mg/L

EC10 or NOEC for freshwater algae:

0.13 mg/L

Additional information

The influence of the test material on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata was tested according to OECD 201 (2006) under static and GLP conditions. Eight different concentrations of the test substance between nominal 0.006 and 10.000 mg per litre nutrient medium, spaced by a factor of about 3.0, were tested against one concurrent negative control (nutrient medium only). Three replicates were used for each test culture and six replicates for the control culture. The cell count of the algae was about 10000 cells/mL at the start of the exposure in each vessel. In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the beginning and at the end of the incubation period. Visual observations on the appearance of the algal cultures were made each time when the cell densities were determined. Possible test substance effects were determined by comparison of the yield and of the growth rates. At the start and at the end of the experiment, samples of the cultures exposed to the test substance, of the control cultures and also of the blank with the highest concentration of the test substance, but no algae, were taken and analysed in duplicate by HPLC. The geometric mean concentrations of the test substance were determined to be 0.01, 0.02, 0.05, 0.13, 0.38, 0.79, 2.85, 8.48 and 8.90 mg/L. The test substance concentrations have not been maintained within 80 % of the nominal concentrations throughout the duration of the test. Therefore the presentation of the growth curves and the calculation of the effective concentrations were based on the mean measured test substance concentrations. No precipitation of test substance was noted during the incubation period. The test media containing the three highest test substance concentrations were coloured light yellow during the first 48 hours of exposure. At the end of the test light yellow colouration was only observed in the two highest test media. All other test and control media were colourless throughout the exposure period. A reference test with potassium dichromate was conducted to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg/L, respectively. These results establish the reliability of the test procedures for this kind of study type. Under the given conditions of this study the 72 h ErC50 value of the test substance was determined to be 1.05 mg/L and the NOEC was determined to be 0.13 mg/L.