1-imino-1H-isoindol-3-amine

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:

2016-12-15 to 2017-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- sampling method: analytical samples were taken in the main at 0h (initial value), after 24h, 48h and 72h from aged solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Stock solution: 18.8 mg of test item was mixed with test AAP-medium and homogenised by shaking for 5 minutes of ultrasonication; Final volume is 1000mL
- Dilutions of stock solution: 3 dilutions were made from the stock solution with test AAP-medium, to reach a final volume of 500mL per dilution
- Controls: test water without addition of the test item

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No. 61.81 SAG
- Source: available in study report
- Age of inoculum (at test initiation): 3 to 4 days old pre-culture

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Microscopic evaluation of the cells at the end of exposure (morphological appearance check)

Test temperature:

22.4 – 23.0 °C

pH:

pH range of control (0h-72h): 7.28 - 7.84
pH range of samples (0h-72h): 7.34 - 7.91

Nominal and measured concentrations:

Nominal concentrations tested (spaced by a factor of 2.5): 18.8, 7.52, 3.01, 1.20, 0.481 mg/L and a control
Measured concentrations (geometric mean concentrations): 6.54, 2.56, 1.03, 0.373, 0.152 mg/L and n.d.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 100mL volume with aluminium caps filled with 50mL test solution
- Initial cells density: 0.5 x 10^4 cells/mL
- Control end cells density: 33.22 x 10^4 cells/mL
- No. of organisms per vessel: 0.5 x 10^4 cells/mL, employed for the individual replicates
- No. of treatment groups: 6 (5 dose rates of the test item and a control)
- No. of replicates: 3 for each test item concentration and 6 for the control

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD AAP-medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination from the side
- Light intensity: 88.8 µE/m2 s

EFFECT PARAMETERS MEASURED: influence of test item on yield and growth rate of organisms

RANGE-FINDING STUDY
- Test concentrations: Control, 100, 10.0, 1.00, 0.100 and 0.0100 mg/L.
- Results used to determine the conditions for the definitive study: after 72 h at termination of the test no inhibition of growth rate or yield was observed for concentrations up to and including 1.00 mg/L. For higher test item concentrations: inhibition of growth rate peaked (reached the highest point) at 142.4 % at a nominal concentration of 100 mg/L and the inhibition of yield peaked at 102.0 % at a nominal concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate tested at least twice a year

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.841 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.:

0.981 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.636 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.746 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.373 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and Yield inhibition

Details on results:

- Exponential growth in the control (for algal test): yes
- Morphology of algae cells: the cells were considered normal for the control and up to and including a nominal test item concentration of 3.01 mg/L. No cells were observed at 7.52 and 18.8 mg/L nominal test item concentration.

Results with reference substance (positive control):

- Results with reference substance valid? yes, the EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.
- Test rates: static main test with nominal concentrations of 0.0512, 0.128, 0.320, 0.800 and 2 mg/L and control
- Conclusions: significant inhibitory effects were determined for all parameters at test item concentrations of 0.800 mg/L (nominal) and above
- EC50 (growth rate): 1.64 mg/L (nominal); EC50 (yield inhibition): 0.976 mg/L (nominal)
- EC10 (growth rate): 0.695 mg/L (nominal); EC10 (yield inhibition): 0.354 mg/L (nominal)

Results non-GLP dose range finding test:

Concentration (mg/L)	% inhibition after 72h
	Growth rate	Yield
Control	0	0
0.01	-7.5	-29.3
0.1	-8.7	-37.1
1	-6.3	-23.8
10	123.5	101.5
100	142.4	102

Biological results main study:

Percentage inhibition of growth rate:

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.481	-1.8	-1.8	1.3
1.20	-10.3	-3.6	-0.2
3.01	14.7	16.7	20.3
7.52	36.4	79.9	83.8
18.8	107.9	109.9	132.3

Average cell number for each sampling time and concentration

Conc.	Average cell numbers/mL [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.52	1.50	6.34	33.22
0.481	0.52	1.49	6.55	31.43
1.20	0.52	1.62	6.94	33.74
3.01	0.52	1.25	4.18	14.35
7.52	0.52	1.00	0.87	1.04
18.8	0.52	0.48	0.41	0.14

Analytical results: determination of test item concentrations

Nominal concentration (mg/L)	Sampling	Measured concentration (mg/L)	% of nominal	Geometric mean (mg/L)
Control	0h fresh	n.d
	24h aged	n.d
	48h aged	n.d
	72h aged	n.d
0.481	0h fresh	0.436	91	0.152
	24h aged	0.215	45
	48h aged	0.107	22
	72h aged	0.0507	11
1.2	0h fresh	1.08	90	0.373
	24h aged	0.538	45
	48h aged	0.275	23
	72h aged	0.122	10
3.01	0h fresh	2.89	96	1.03
	24h aged	1.51	50
	48h aged	0.723	24
	72h aged	0.368	12
7.52	0h fresh	7.51	100	2.56
	24h aged	3.72	49
	48h aged	1.74	23
	72h aged	0.879	12
18.8	0h fresh	19.3	103	6.54
	24h aged	9.83	52
	48h aged	4.67	25
	72h aged	2.09	11

Validity criteria fulfilled:

yes

Conclusions:

After an exposure of 72 h, the test item was found to inhibit the growth of Pseudokirchneriella subcapitata. The reported EC50-values (based on measured concentrations, geom. mean) are 1.56 mg/L for growth rate inhibition and 0.981 mg/L when yield inhibition was studied. The 72-hour EC10-values (based on measured concentrations, geom. mean) for inhibition of growth rate and yield were 0.841 and 0.636 mg/L, respectively.

Executive summary:

For this endpoint, one study is available in which the effect of the test item on growth of freshwater green algae Pseudokirchneriella subcapitata after 72 hours exposure was assessed according to OECD 201. The organisms were exposed over a period of 72 hours to 5 concentrations of the test item (nominal concentrations of: 18.8, 7.52, 3.01, 1.20, 0.481 mg/L and control; corresponding to measured concentrations (geom. mean): 6.54, 2.56, 1.03, 0.373, 0.152 mg/L and n.d.).

The test item was found to inhibit the growth of Pseudokirchneriella subcapitata. The 72-hour EC50-values (based on measured concentrations) for both inhibition of growth rate (ErC50) and yield (EyC50) were 1.56 and 0.981 mg/L. The EC10-values (based on measured concentrations) for inhibition of growth rate and yield after 72 hours were 0.841 and 0.636 mg/L, respectively.

Validity criteria were fulfilled with this test set-up.

Description of key information

For this endpoint, one study is available in which the effect of the test item on growth of freshwater green algae Pseudokirchneriella subcapitata after 72 hours exposure was assessed according to OECD 201.

The 72-hour EC50-values (geometric mean, measured concentrations) for both inhibition of growth rate (ErC50) and yield (EyC50) were 1.56 and 0.981 mg/L. The EC10-values (geometric mean, measured concentrations) for inhibition of growth rate and yield after 72 hours were 0.841 and 0.636 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.56 mg/L

EC10 or NOEC for freshwater algae:

0.841 mg/L

Additional information

The organisms were exposed over a period of 72 hours to 5 concentrations of the test item (nominal concentrations of: 18.8, 7.52, 3.01, 1.20, 0.481 mg/L and control. This corresponds to measured concentrations (geom. mean): 6.54, 2.56, 1.03, 0.373, 0.152 mg/L and n.d.).

Validity criteria were fulfilled with this test set-up.

The test item was found to inhibit the growth of Pseudokirchneriella subcapitata.