Tetrachloro-p-benzoquinone

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-06 to 2009-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to current OECD-Guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrachloro-p-benzoquinone

EC Number:

204-274-4

EC Name:

Tetrachloro-p-benzoquinone

Cas Number:

118-75-2

Molecular formula:

C6Cl4O2

IUPAC Name:

2,3,5,6-tetrachloro-1,4-benzoquinone

Details on test material:

- Name of test material (as cited in study report): Tetrachloro-p-benzoquinone
- Molecular formula: C6Cl4O2
- Molecular weight: 245.88 g/mol
- Physical state: Solid, yellow crystalline powder
-

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: At test start two samples of the saturated solution, before and after adjusting the pH value, were prepared and analysed. Separate samples without algae were prepared at test start. At end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* of 40 mg/L was prepared with dilution water. The pH value of the dilution water was adjusted to
pH 6 ± 0.2 (by addition of 1 N NaOH and HCl) because the substance is stable under these conditions. The dispersion was shaken with 20 rpm for 24 h at ambient temperature and filtrated with 0.45 µm RC filter. Prior to preparing the dilution levels the saturated solution was adjusted to pH 8.1 ± 0.2 by addition of 1 M NaOH.
- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor 2: 1 - 3.2 - 10 - 32 and 100 % of the saturated solution corresponding to initially measured concentrations of 0.0115 – 0.0364 – 0.115 – 0.373 – 1.23 mg/L.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Initially measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
1.23 7.98 8.08
0.373 8.12 8.35
0.115 8.08 8.48
0.03641) 7.92 8.57
0.01151) 8.01 8.46
Control 8.07 8.73

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on material and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1004482 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)

Dilution level out of the saturated solution
[%] Rate Related Inhibition [%] Yield Inhibition[%]
100 9 39
10 3 15
1 1 7

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was =0.05.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % were marked biological significant.

Any other information on results incl. tables

NOEC, LOEC and EC - values of Tetrachloro-p-benzoquinone(0-72 h)

                   based on initially measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	1.23
LOEC	> 1.23
ErC10	> 1.23
ErC20	> 1.23
ErC50	> 1.23
	Inhibition of Yield
NOEC	0.373
LOEC	1.23
EyC10	0.373 EC10 1.23
EyC20	0.373 EC20 1.23
EyC50	> 1.23

Cell Densities

	Initially measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
1.23		1		5074		22930	117931	607680
		2		5074		20580	120382	598414
		3		5074		20027	132592	629643
		Mean		5074		21179	123635	611912
0.373		1		5074		28674	263669	1175526
		2		5074		32524	244559	1128716
		3		5074		31786	268319	1163951
		Mean		5074		30995	258849	1156064
0.115		1		5074		36919	235281	1040267
		2		5074		35078	271636	1238674
		3		5074		33077	256490	1012813
		Mean		5074		35025	254469	1097251
0.0364^1)		1		5074		31630	174666	970460
		2		5074		38264	269024	1130606
		3		5074		35693	247892	884364
		Mean		5074		35196	230527	995143
0.0115^1)		1		5074		37083	212350	1113415
		2		5074		31593	253436	1033444
		3		5074		31043	212399	818813
		Mean		5074		33240	226062	988557
Control		1		5074		40215	233768	1174037
		2		5074		34188	193051	928681
		3		5074		36587	209992	886258
		4		5074		36115	241693	1130983
		5		5074		36082	180509	965495
		6		5074		32511	230763	941436
		Mean		5074		35950	214963	1004482

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Initially measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.23	1		1.60	9.41		602606	39.7
	2		1.59	9.70		593340	40.6
	3		1.61	8.74		624569	37.5
	Mean	(+)*	1.60	9.28	(+)	606838	39.3
0.373	1		1.82	-3.08		1170452	-17.1
	2		1.80	-2.31		1123642	-12.4
	3		1.81	-2.90		1158877	-16.0
	Mean	(-)	1.81	-2.76	(-)	1150990	-15.2
0.115	1		1.77	-0.77		1035193	-3.58
	2		1.83	-4.07		1233600	-23.4
	3		1.77	-0.26		1007739	-0.83
	Mean	(-)	1.79	-1.70	(-)	1092177	-9.28
0.0364^1)	1		1.75	0.55		965386	3.40
	2		1.80	-2.34		1125532	-12.6
	3		1.72	2.31		879290	12.0
	Mean	(-)	1.76	0.17	(-)	990069	0.93
0.0115^1)	1		1.80	-2.05		1108341	-10.9
	2		1.77	-0.64		1028370	-2.90
	3		1.70	3.76		813739	18.6
	Mean	(-)	1.76	0.36	(-)	983483	1.59
Control	1		1.82			1168963
	2		1.74			923607
	3		1.72			881184
	4		1.80			1125909
	5		1.75			960421
	6		1.74			936362
	Mean	(-)	1.76		(-)	999408

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	2.07	1.76	1.61	1.82	0.233	12.8	13.1
	2	1.91	1.73	1.57	1.74	0.169	9.71
	3	1.98	1.75	1.44	1.72	0.269	15.6
	4	1.96	1.90	1.54	1.80	0.227	12.6
	5	1.96	1.61	1.68	1.75	0.187	10.7
	6	1.86	1.96	1.41	1.74	0.295	16.9
				Mean	1.76
				SD ±	0.04
				CV [%]	2.17

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Tetrachloro-p-benzoquinone was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 1.23 and 0.373 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were > 1.23 and 1.23 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were > 1.23 mg/L, respectively which represents the maximum saturation under test conditions.
All effect levels are given based on initially measured concentrations of Tetrachloro-p-benzoquinone.

Executive summary:

The toxicity of Tetrachloro-p-benzoquinone(batch number INCB 5070661) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 , from October 06 to 09, 2009. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h. The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary test, a definitive test with the saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 1 - 3.2 - 10 - 32 - 100 % of the saturated solution corresponding to initially measured test item concentrations of 0.0115 – 0.0364 – 0.115 – 0.373 – 1.23 mg/L. The two lowest concentrations were calculated based on the results of the third lowest test item concentration with the dilution factor of √10. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Tetrachloro-p-benzoquinonewere analysed at all concentration levels after 0 and 72 h via HPLC analysis. The two lowest dilution levels were below the LOQ of   0.075 mg/L at test start. The measured concentrations for the two highest concentrations of Tetrachloro-p-benzoquinoneat test end were in the range of 31 – 67 % of the initially measured values. Concentrations of the lower concentrations were less than the LOQ of 0.075 mg/L. All effect values are given based on initially measured concentrations of the test item.

Table 1:    NOEC, LOEC and EC - values of Tetrachloro-p-benzoquinone(0-72 h)

                   based on initially measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	1.23
LOEC	> 1.23
ErC10	> 1.23
ErC20	> 1.23
ErC50	> 1.23
	Inhibition of Yield
NOEC	0.373
LOEC	1.23
EyC10	0.373 EC10 1.23
EyC20	0.373 EC20 1.23
EyC50	> 1.23