4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

none

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from the control and each test group (replicates R1 - R3 pooled) on day 0 (fresh media) and on day and 7 (old media) for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0% v/v saturated solution.

Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give the required test concentration of 100% v/v saturated solution.

Test organisms (species):

Lemna minor

Details on test organisms:

The test was carried out using Lemna minor. A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Post exposure observation period:

None

Hardness:

None

Test temperature:

The temperature and light intensity in the incubator were recorded daily. Temperature was maintained at 24 ± 2 ºC throughout the test.

pH:

The pH of each control and test flask was recorded on Day 0 and Day 7. The temperature and light intensity in the incubator were recorded daily.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Range-finding test: Nominal test concentrations of 1.0, 10 and 100% v/v saturated solution
Definitive test: : 6.25, 12.5, 25, 50 and 100% v/v saturated solution

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 7 days.

The test was conducted in glass conical flasks (500 mL). Two replicate flasks were prepared for each control and test concentration.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 2 ºC under continuous illumination (intensity approximately 7000 lux) for 7 days.

On Days 2 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 2, 5 and 7.

In order to determine the stability of the test item under test conditions a sample of each test concentration was taken for chemical analysis on Day 0 (fresh media) and Day 5 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.

The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day 7.

Exposure Conditions
As in the range-finding test glass conical flasks were used. Three flasks each containing 250 mL of solution were prepared for the control and six flasks for the treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Each control and test flask was inoculated with 3 colonies of Lemna minor (total 9 fronds). The flasks were then incubated at 24 ± 2 ºC under constant illumination (intensity approximately 7000 lux) for 7 days.

A static testing regime was employed.

Evaluations
Test Organism Observations
The number of fronds present in each test and control culture was recorded on days 0, 2, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.

In addition the dry weight of the fronds in each control and treatment group was determined on day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by blotting the colonies dry and drying at 60 °C.

Data Analysis
Doubling Time
The doubling time (Td) of frond number, and hence performance of the test against the validation criterion of doubling time, was calculated for the control vessels using the following formula:

Td = ln 2/µ

Where:
µ = the average specific growth rate for the control vessels.


Response Variables
In order that the test results are acceptable to regulatory authorities worldwide, the effects on the growth of Lemna were evaluated using both response variables (a) and (b) described below:

a) Average specific growth rate: this response variable was calculated on the basis of changes in the logarithms of frond numbers, and in addition, on the basis of changes in the logarithms of dry weight over time (expressed per day) in the controls and each treatment group.

b) Yield: this response variable was calculated on the basis of changes in frond number, and in addition, on the basis of changes in dry weight in the controls and in each treatment group until the end of the test.

It should be noted that toxicity values calculated using these two response variables are not comparable and the difference must be recognized when using the results of the test. ECx values based upon average specific growth rate (ErCx) will be higher than results based upon yield (EyCx) under the test conditions of this Guideline, due to the underlying mathematics of the respective approaches. This is not to be interpreted as a difference in sensitivity between the response variables: simply the values are different mathematically. The concept of average specific growth rate is based upon the general exponential growth pattern of Lemna in non-limited cultures. Toxicity is estimated on the basis of the effects on the growth rate without being dependent on the absolute level of the specific growth rate of the control, slope of the concentration-response curve or on test duration. Results based upon yield are dependent upon all these variables.

Average Specific Growth Rate
The average specific growth rate for a specific period was calculated as the logarithmic increase in the growth variables -frond numbers and dry weight - using the formula below for each replicate of the control and treatment groups:

µi-j = (1n(Nj) – 1n(Ni)) / t

Where:
µi-j =Average specific growth rate from time i to j
Ni = number of fronds or dry weight in the test or control vessel at time i
Nj = number of fronds or dry weight in the test or control vessel at time j
t = time period from i to j

For each control and treatment group the mean value for growth rate along with the standard deviation was calculated.

Percentage inhibition of growth rate (Ir) was calculated as follows:

%Ir = ((µc - µt) / µc) x 100

Where:
% I = percentage inhibition of average specific growth rate
µc = mean value of µ for the control
µt = mean value of µ for the treatment group

Yield
Effects on yield were determined on the basis of frond numbers and dry weight present in each test vessel at the start and end of the test. For dry weight, the starting biomass was determined on the basis of a sample of fronds taken from the same batch used to inoculate the test vessels. For each test concentration and control, the mean value for yield along with variance estimates was calculated.

Percentage inhibition in yield (I ) was calculated for each treatment group as follows:

%Iy = ((bc – bt) / bc) x 100

Where:
% Iy = percentage reduction in yield
bc = final biomass minus starting biomass for the control group
bt = final biomass minus starting biomass for the treatment group


Determination of ECx Values
Percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.

It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: Average specific growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight average sepcific growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Dry weight yield

Details on results:

Range-finding test
The results showed no significant effect on growth at the test concentration of 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.

Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the test preparations on Days 0, 5 and 7 showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.025 mg/L were obtained. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Definitive Test
Growth Data Based on Frond Number
The following results based on inhibition of average specific growth rate and yield were determined from the frond number data:

Average Specific Growth Rate
ErC10 (frond number) = >100% v/v saturated solution
ErC20 (frond number) = >100% v/v saturated solution
ErC50 (frond number) = >100% v/v saturated solution

Where:

ErCx = the test concentration that reduced average specific growth rate by x%.

Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5, 25, 50 and 100% v/v saturated solution test concentrations (P 0.05), and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 100% v/v saturated solution.


Yield
EyC10 (frond number) = >100% v/v saturated solution
EyC20 (frond number) = >100% v/v saturated solution
EyC50 (frond number) = >100% v/v saturated solution

Where:

EyCx = the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5, 25, 50 and 100% v/v saturated solution test concentrations (P 0.05), and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition ofyield calculated from frond numbers was 100% v/v saturated solution.


Growth Data Based on Dry Weight
Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:


Average Specific Growth Rate
ErC10 (dry weight) = >100% v/v saturated solution
ErC20 (dry weight) = >100% v/v saturated solution
ErC50 (dry weight) = >100% v/v saturated solution

Where:

ErCx = the test concentration that reduced average specific growth rate by x%.

Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control 6.25, 12.5, 25, 50 and 100% v/v saturated solution test concentrations (P 0.05),) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 100% v/v saturated solution.


Yield
EyC10 (dry weight) = >100% v/v saturated solution
EyC20 (dry weight) = >100% v/v saturated solution
EyC50 (dry weight) = >100% v/v saturated solution

Where:

EyCx = the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5, 25, 50 and 100% v/v saturated solution test concentrations (P 0.05), and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 100% v/v saturated solution.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41403075) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Lemna minor to the reference item gave the following results:

The average specific growth rate (frond number) EC50 was 3.4 mg/L with 95% confidence limits of 3.1 - 3.6 mg/L. The NOEC was 2.5 mg/L and the LOEC was 5.0 mg/L.
The average specific growth rate (dry weight) EC50 was 3.4 mg/L with 95% confidence limits of 3.2 - 3.7 mg/L. The NOEC was 1.25 mg/L and the LOEC was 2.5 mg/L.
The yield (frond number) EC50 was 2.6 mg/L with 95% confidence limits of 2.4 - 2.9 mg/L. The NOEC was 2.5 mg/L and the LOEC was 5.0 mg/L.
The yield (dry weight) EC50 was 2.7 mg/L with 95% confidence limits of 2.5 - 2.9 mg/L. The NOEC was 1.25 mg/L and the LOEC was 2.5 mg/L.

The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981), and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955), were carried out on the average specific growth rate and yield data at 7 days for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

Verification of Test Concentrations

Chemical analysis of the 100 % v/v saturated solution on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0025 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Validation Criteria

The following data show that the doubling time of the control cultures was 1.79 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:

Mean frond number in control cultures at day 0: 9

Mean frond number in control cultures at day 7: 93

Frond Numbers and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)		Number of Fronds				Average Specific Growth Rate		Yield
		Day 0	Day 2	Day 5	Day 7	(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	R1	9	16	43	91	0.331	-	82	-
	R2	9	19	44	90	0.329		81
	Mean	9	18	44	91	0.330		82
1.0	R1	9	16	46	96	0.338	[3]	87	[7]
	R2	9	20	48	98	0.341		89
	Mean	9	18	47	97	0.340		88
10	R1	9	16	40	89	0.327	[2]	80	[5]
	R2	9	19	47	101	0.345		92
	Mean	9	18	44	95	0.336		86
100	R1	9	14	36	72	0.297	7	63	16
	R2	9	12	36	83	0.317		74
	Mean	9	13	36	78	0.307		69

R₁– R₂= Replicates 1 and 2

[Increase in growth as compared to the controls]

Frond Numbers from the Definitive Test

Nominal Concentration (%v/v Saturated Solution)		Numberof Fronds
		Day 0	Day 3	Day 5	Day 7
Control	R1	9	22	46	95
	R2	9	22	48	101
	R3	9	19	40	83
	Mean	9	21	45	93
6.25	R1	9	25	52	108
	R2	9	23	48	97
	R3	9	23	52	96
	Mean	9	24	51	100
12.5	R1	9	22	44	102
	R2	9	21	44	96
	R3	9	21	41	96
	Mean	9	21	43	98
25	R1	9	25	51	97
	R2	9	18	40	77
	R3	9	22	44	95
	Mean	9	22	45	90
50	R1	9	23	44	95
	R2	9	21	41	90
	R3	9	21	41	88
	Mean	9	22	42	91
100	R1	9	21	40	93
	R2	9	24	55	103
	R3	9	20	39	83
	Mean	9	22	45	93

R1- R3 = Replicates 1 to 3

Table: Inhibition of Average Specific Growth Rate and Yield Based on Frond Numbers

Nominal Concentration (%v/v Saturated Solution)		Average Specific Growth Rate		Yield
		(0 -7 Day)	% Inhibition	(0-7 Day)	% Inhibition
Control	Mean	0.333	-	84	-
	SD	0.014		9
6.25	Mean	0.344	[3]	91	[8]
	SD	0.009		7
12.5	Mean	0.341	[2]	89	[6]
	SD	0.005		3
25	Mean	0.328	2	81	4
	SD	0.018		11
50	Mean	0.331	1	82	2
	SD	0.006		4
100	Mean	0.333	0	84	0
	SD	0.016		10

SD - Standard deviation

[Increase in growth compared to controls]

Table: Dry Weights from the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		Dry Weight(mg)
		Day 0*	Day 7
Control	R1		9.9
	Rz		10.4
	R3		7.5
	Mean	0.6	9.3
6.25	RI		12.3
	Rz		10.9
	R3		12.2
	Mean	0.6	11.8
12.5	R1		12.4
	R2		9.2
	R3		9.3
	Mean	0.6	10.3
25	R1		12.2
	R2		6.9
	R3		10.9
	Mean	0.6	10.0
50	R1		8.6
	R2		10.0
	R3		10.7
	Mean	0.6	9.8
100	R1		8.5
	R2		11.5
	R3		11.4
	Mean	0.6	10.5

* Mean value determined from 6 replicate weighings

R1 -R3 = Replicates 1 to 3

Table: Inhibition of Average Specific Growth Rate and Yield Based on Dry Weight

Nominal Concentration (% v/v Saturated Solution)		AverageSpecificGrowth Rate		Yield
		(0-7 Day)	% Inhibition	(0-7 Day)	%Inhibition
Control	Mean	0.390	-	8.7	-
	SD	0.025		1.6
6.25	Mean	0.425	[9]	11.2	[29]
	SD	0.010		0.8
12.5	Mean	0.405	[4]	9.7	[11]
	SD	0.024		1.8
25	Mean	0.398	[2]	9.4	[8]
	SD	0.043		2.8
50	Mean	0.398	[2]	9.2	[6]
	SD	0.016		1.1
100	Mean	0.407	[4]	9.9	[4]
	SD	0.025		1.7

SD - Standard deviation

[Increase in growth as compared to the controls]

Table: pH Values in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		Time (Days)
		0	7
Control	R1	7.0	7.9
	R2	7.0	7.9
	R3	6.9	7.9
6.25	R1	6.9	7.9
	R2	6.9	7.9
	R3	6.9	7.9
12.5	R1	6.9	7.9
	R2	6.9	8.0
	R3	6.9	7.9
25	R1	6.9	7.9
	R2	6.9	7.9
	R3	6.9	7.9
50	R1	6.9	7.9
	R2	6.9	7.8
	R3	6.9	7.8
100	R1	7.0	8.1
	R2	7.0	8.1
	R3	7.0	8.0

R1 - R3 = Replicates 1 to 3

Validity criteria fulfilled:

yes

Conclusions:

The average specific growth rate (frond number) EC50 was >100 % v/v saturated solution, while the NOEC was 100 % v/v saturated solution.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna Growth Inhibition Test (March 2006)”.

 .

Information provided by the Sponsor indicated the water solubility of the test item to be less than 2.3 µg/L. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

A preliminary media preparation trial indicated that a saturated solution method of preparation, followed by the removal of any undissolved test item by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded) was most appropriate for this test item.

 

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 2°C. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

 

The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development.

 

Chemical analysis of the 100 % v/v saturated solution test preparation on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0025 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

 

Hence based on the findings of the study, following results were obtained:

The average specific growth rate (frond number) EC50 was > 100 % v/v saturated solution

The average specific growth rate (dry weight) EC50 was > 100 % v/v saturated solution

The yield (frond number) EC50 was > 100 % v/v saturated solution

The yield (dry weight) EC50 was > 100 % v/v saturated solution

The NOEC was 100 % v/v saturated solution.

Description of key information

 The average specific growth rate (frond number) EC50 was > 100 % v/v saturated solution, while the NOEC was 100 % v/v saturated solution. 

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

100 mg/L

Additional information

A key study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 211 “Lemna Growth Inhibition Test (March 2006).

Chemical analysis of the 100% v/v saturated solution test preparation on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.025 mg/L.

Exposure of Lemna minor to the test item gave the following results:

The average specific growth rate (frond number) EC50 was > 100 % v/v saturated solution

The average specific growth rate (dry weight) EC50 was > 100 % v/v saturated solution

The yield (frond number) EC50 was > 100 % v/v saturated solution

The yield (dry weight) EC50 was > 100 % v/v saturated solution

The NOEC was 100 % v/v saturated solution.

 

Therefore, it can be concluded that there were no toxic effects at saturation.