Bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

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:

2014-11-16 to 2015-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD 201 and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution:
The saturated solution (1.00 mg/L test item was weighed out) was prepared with demineralized water two days prior to the start of the exposure (0 h).

Treatment
The dispersion was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded.
The filtration was interrupted for approx. 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded.
After the separation treatment the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered.

Test concentration
The saturated solution was tested in a limit test.


Control:
Six replicates (without test item) were tested under the same test conditions as the test replicates.

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): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 22.0, mean value: 22.8

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 8.00 8.23
Control 8.04 8.28

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- 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: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4847 cells/mL
- Control end cells density: Mean 1388398 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



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, mean value: 70.6



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

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: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Set-up and
Experimental Date 2015-04-14 to
2015-04-17 Valid Range)
(average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.546 0.671 ± 0.413
95 % confidence interval 0.496 - 0.606
Yield Inhibition
EyC50 0.281 0.317 ± 0.139
95 % confidence interval 0.220 - 0.347

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

                  Cell Densities

 

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.00	1	4847	33416	278401	1496698
	2	4847	36562	257504	1470241
	3	4847	32396	254752	1385624
	4	4847	43379	262892	1459946
	5	4847	38665	249076	1237557
	6	4847	35562	225341	1439453
	Mean	4847	36663	254661	1414920
Control	1	4847	32853	286425	1331987
	2	4847	40146	290244	1445563
	3	4847	35980	261371	1458262
	4	4847	30323	263065	1360417
	5	4847	41565	273128	1297688
	6	4847	36067	276188	1436471
	Mean	4847	36156	275070	1388398

 

 

 

   Evaluation after 72 hours

                  Statistically significant differences of growth rates and yield compared to

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

 

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.91	-1		1491851	-8
	2		1.91	-1		1465394	-6
	3		1.89	0		1380777	0
	4		1.90	-1		1455099	-5
	5		1.85	2		1232710	11
	6		1.90	-1		1434606	-4
	Mean	(-)	1.89	0	(-)	1410073	-2
Control	1		1.87			1327140
	2		1.90			1440716
	3		1.90			1453415
	4		1.88			1355570
	5		1.86			1292841
	6		1.90			1431624
	Mean		1.89			1383551

 

 

 

  Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

 

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.91	2.17	1.54	1.87	0.316	16.9	13.4
	2	2.11	1.98	1.61	1.90	0.263	13.9
	3	2.01	1.98	1.72	1.90	0.159	8.4
	4	1.83	2.16	1.64	1.88	0.262	13.9
	5	2.15	1.88	1.56	1.86	0.296	15.9
	6	2.01	2.04	1.65	1.90	0.216	11.4
				Mean	1.89
				SD ±	0.02
				CV [%]	0.86

CV= Coefficient of variation

SD= Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values of the test item for both inhibition of growth rate and inhibition of yield after 72 hours were = 1.00 mg/L, respectively. The EC50-values of the test item for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 1.00 mg/L, respectively.
All effect levels are given based on the nominal concentration of the test item.

Executive summary:

The toxicity of the test item (batch number:USEA016274) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) . The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4847 cells/mL. A saturated solution with a nominal concentration of 1.00 mg/L was assessed. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

All effect values of the test item are given based on the nominal concentration.

 

 

Table1:  NOEC, LOEC and EC-values of the test item (0-72 hours)

                  based on nominal test item concentrations [mg/L]

                               

	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield Nominal test item concentration [mg/L]
NOEC	1.00
LOEC	> 1.00
EyC10	< 1.00
EyC20	> 1.00
EyC50	> 1.00

 

Description of key information

The toxicity of the test item to the unicellular freshwater green alga (Desmodesmus subspicatus) was determined according to OECD 201 (2011). The study was conducted under static conditions with a saturated solution prepared from a nominal concentration of 1.00 mg/L of the test item. In this study the test item did not cause any inhibiting effects to Desmodesmus subspicatus after 72 h.

The No Observed Effect Concentration (NOEC) was assessed as 1.00 mg/L and the EC₅₀ was laid down as > 1.00 mg/L (based on the nominal concentration)