N-ethylpropylamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29-Nov-21 to 2-Dec-21

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:

the requirements of OECD GD23 were fulfilled since the test substance ionized a certain adsorption potential could not be excluded. All validity criteria were met

Deviations:

yes

Remarks:

Modified exposure media, closed test system, and reduced inoculation density. Additional test showed no effect of theses deviation on the results of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temp.
- Stability and homogeneity during storage: yes, guaranteed by the sponsor
- Reactivity of the test material with the incubation material used: highly adsorptive to glass ware

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: homogenized by gently shaking

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
0h: uninoculated, replicate 7 from each test group
72h: combined inoculated replicates from each test group and from additional uninoculated replicate
- Sample storage conditions before analysis: at the freezer (at approx. -20°C) until transported
(see table 1 for sampling scheme)

Vehicle:

no

Remarks:

only OECD medium was used

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct pipetting of the test substance into the test medium, stirring for 10 min at approx 800 rpm at room temperature
- Controls: visually inspection for undissolved substance and Tyndall effect was measured (negative)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):
- Other relevant information: pH was adjusted when outside the +- 0.5 of the control with HCl or NaOH
- Test concentration separation factor: <= 3.2

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Korshikov (SAG 61.81)
- Source (laboratory, culture collection): Collection of algal cultures, University of Göttingen, Germany
- Age of inoculum (at test initiation): 3 days

PREPARATION OF INOCULUM
- Growth phase of used culture: exponential
- cells control: microscopicalli for normal morphology

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.2 – 23.4 °C

pH:

see table 2

Nominal and measured concentrations:

nominal: 0 (control), 3.2, 10, 32, 100, and 320 mg/L
mean measured: (control), 3.10, 6.69, 32.63, 101.12, and 307.75 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks, closed with glass plugs (nominal volume 250 mL)
- Type: closed
- Material, size, headspace, fill volume: Approx. 300 mL (Erlenmeyer flasks were completely filled, no headspace)
- Aeration: stirring, continuous (approx. 130 rpm) or twice daily by hand
- Initial cells density: 0.3 x 104 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 additional replicate per test group for initial concentration control analysis only
- 1 additional uninoculated (without algae) replicate per test group for background fluorescence correction and to determine the influence of the test design on the test substance stability

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes with HCl or NaOH
- Photoperiod: permanent illumination (test vessels were rearranged daily)
- Light intensity and quality: 5375 - 5875 lux (mean: 5611 lux, coefficient of variation: 2.85%) at a wavelength of 400 - 700 nm

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: fluorometric; excitation / emission wavelength = 430 / 670 nm; excitation / emission bandwidth = 20 / 25 nm; flashes = 5; integration time = 20 μs; shaking duration = 15s;
shaking amplitude = 6 mm
- Measurement intervals: 0, 24, 48 and 72 h

RANGE FINDING STUDY
- Results used to determine the conditions for the definitive study:
EC50 > 120 mg/L

Reference substance (positive control):

yes

Remarks:

Name of reference substance: 3,5 Dichlorphenol Reference substance No.: 02/0308-8 Content: 99.7% Batch identification: 10173456 CAS No.: 591-35-5 Expiry date: 20 Jun 2022

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 307.75 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.:

20.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 3.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

An additional reference toxicant test was conducted with the same method modifications described in this study (modified exposure media, closed test system, and reduced inoculation density) todetermine any effect on algal response to toxic stress.
T he ErC50 (72 h) of the control substance 3,5 Dichlorphenol was 3.05 mg/L (Date of the last control experiment: 31 Aug 2021, project number: 60E0308/02E018).
These results indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results
of this study.
No remarkable observations during the test period regarding the algal morphology were made.
There was no evidence of undissolved test material during the test period.

Table 3: Analytical results for the measured concnetrations: Mean measured concentrations (geometric mean) are based on 0-h
uninoculated (-algae=abiotic), and 72-h inoculated (+algae=biotic) samples. 72-h uninoculated
samples were excluded from the calculations.

Nominal Concentration [mg/L]	Sample Number	Sampling Time (hours)	Algae +a=biotic ‒a=abiotic	Measured Concentrations [mg/L]         % Nominal
0 (Control)	1	0	‒a	<LOQ**	‒
	11	72	+a	<LOQ**	‒
	21	72	‒a	<LOQ**	‒
geometric mean	‒	‒	‒	‒	‒
3.2	2	0	‒a	3.06	95.5%
	12	72	+a	3.14	98.1%
	22*	72	‒a	3.17	98.9%
geometric mean	‒	‒	‒	3.10	96.8%
10	3	0	‒a	6.74	67.4%
	3b	0	‒a	6.16	61.6%
	3c	0	‒a	6.46	64.6%
	13	72	+a	6.94	69.4%
	23*	72	‒a	6.99	69.9%
geometric mean	‒	‒	‒	6.69	66.9%
32	4	0	‒a	31.94	99.8%
	14	72	+a	33.33	104.2%
	24*	72	‒a	34.58	108.1%
geometric mean	‒	‒	‒	32.63	102.0%
100	5	0	‒a	98.65	98.6%
	15	72	+a	103.66	103.7%
	25*	72	‒a	101.42	101.4%
geometric mean	‒	‒	‒	101.12	101.1%
320	6	0	‒a	317.34	99.2%
	16	72	+a	298.45	93.3%
	26*	72	‒a	315.81	98.7%
geometric mean	‒	‒	‒	307.75	96.2%

Table 4: Growth inhibition.72-hour results of the algal growth inhibition test including effect concentrations based on nominal and mean measured concentrations

Nominal concentration [mg/L]	Mean Cell Density (cells/mL) x 104 at 72 hours	Mean yield at 72 hours	Mean specific growth rate at 72 hours
0 (control)	107.80	107.57	2.05
3.2	83.52	83.29	1.97
10	71.78	71.54	1.91
32	47.27	47.02	1.75
100	40.25	40.02	1.73
320	14.27	14.01	1.34

Table 5: Effect concentrations (mg/L) after 72 h based on nominal concentrations

Algal Yield [mg/L]		Algal Growth Rate [mg/L]
EyC10 confidence limits 95%:	n.d. (< 3.2)* n.d.	ErC10 confidence limits 95%:	22.04 13.40 – 36.28
EyC20 confidence limits 95%:	2.88 1.95 – 4.26	ErC20 confidence limits 95%:	91.24 68.57 – 121.41
EyC50 confidence limits 95%:	26.74 21.78 – 32.82	ErC50 confidence limits 95%:	n.d. (> 320)* n.d.
NOEyC	< 3.2	NOErC	< 3.2
LOEyC	3.2	LOErC	3.2

n.d.: not determined due to mathematical reasons or inappropriate data         *: based on judgement of the study director

 

Table 6: Effect concentrations (mg/L) after 72 h based on measured (geometric mean) concentrations

Algal Yield [mg/L]		Algal Growth Rate [mg/L]
EyC10 confidence limits 95%:	n.d. (< 3.1)* n.d.	ErC10 confidence limits 95%:	20.18 11.63 – 35.00
EyC20 confidence limits 95%:	2.30 1.53 – 3.46	ErC20 confidence limits 95%:	87.65 64.28 – 119.51
EyC50 confidence limits 95%:	24.13 19.44 – 29.95	ErC50 confidence limits 95%:	n.d. (> 307.75)* n.d.
NOEyC	< 3.1	NOErC	< 3.1
LOEyC	3.1	LOErC	3.1

n.d.: not determined due to mathematical reasons or inappropriate data         *: based on judgement of the study director

 

Validity criteria fulfilled:

yes

Conclusions:

There is a high probability that the product is not acutely harmful to aquatic aquatic algae (EC50 (72 h) > 307.5 mg/L, measured) .

Executive summary:

In a 72-hour algal growth study, cultures of Raphidocelis subspicatus were exposed to the test item at mean measured concentrations of 0 (control), 3.10, 6.69, 32.63, 101.12, and 307.75 mg/L under static conditions in accordance with the OECD 201 guideline. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. The following statistical effect concentrations were obtained after 72 hours of exposure, based on mean measured concentrations (geometric mean): EC50 (72h) > 307.75 mg/L, EC10 (72h) = 20.15 mg/L and NOEC < 3.1 mg/L. Since the measured concentrations of the 10 mg/L treatment deviated markedly from the nominal concentrations, the effect concentrations based on the mean measured concentrations should be preferably used for the evaluation of the test substance.

The purpose of this study is to determine the effects of the test substance on algal growth and inhibition of growth is the primary response variable. Additionally, yield is also evaluated as a response variable to satisfy regulatory requirements in some countries. ECx values based upon average specific growth rate (ErCx) will generally be higher than results based upon yield (EyCx) according to the test design of the OECD 201 Guideline. The guideline acknowledges this difference and recommends that it should not be interpreted as a difference in sensitivity between the two response variables, simply that the values are different due to mathematical

bias. Furthermore, the guideline states that the use of average specific growth rate for estimating toxicity is scientifically preferred.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Description of key information

The test item is acutely not harmful to aquatic algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

307.75 mg/L

EC10 or NOEC for freshwater algae:

20.15 mg/L

Additional information

An experimental study was conducted to assess the acute toxicity of the test item to aquatic algae. In a 72-hour algal growth study, cultures of Raphidocelis subspicatus were exposed to the test item at mean measured concentrations of 0 (control), 3.10, 6.69, 32.63, 101.12, and 307.75 mg/L under static conditions in accordance with the OECD 201 guideline and the requirements of GD23 since the test substance ionized and a certain adsorption potential cannot be excluded. Anyways, the test item concentrations were constant throughout the test and the results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. After 72 h of exposure the statistical effect concentrations EC50 (72h) > 307.75 mg/L, EC10 (72h) = 20.15 mg/L and NOEC < 3.1 mg/L were obtained, based on mean measured concentrations (geometric mean). Since the measured concentrations of the 10 mg/L treatment deviated markedly from the nominal concentrations, the effect concentrations based on the mean measured concentrations were preferably used for the evaluation of the test substance. Due to scientifically preferences the average of the specific growth rate was used. (BASF, 2022)

In conclusion the test item is probably not harmful to aquatic algae.