Nonanoic acid

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:

2020-04-27 to 2020-08-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted March 23rd, 2006; corrected July 28th, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentrations of the test item in the water phase were measured by chemical analysis using GC-MS at test initiation, after 24 hours, 48 hours and at test termination.
Samples were taken from all test concentrations and controls at the beginning of the test but prior to the addition of the algae. This resulted in two sets of samples containing 6 samples each. One set was analysed while one set served as retain sample set. After 24 and 48 hours, samples were taken from additionally prepared replicates (including algae) per test concentration and controls and at test termination samples were taken from representative replicates (2 sets of 6 samples each).

Vehicle:

no

Details on test solutions:

Since the test item is of low water solubility, the highest test concentration was prepared comparable to a Water Accommodated Fraction of the test item, as described in the OECD guideline No. 23, to achieve the maximum solubility of the test item.
209.82 mg of the test item (corresponding to a nominal concentration of 90.5 mg a.s./L considering a purity of 98.8% and a density of 0.905) was transferred into a sterile brown glass flask (acetone washed and sterilised) that had a bottom draining port. A volume of 2.29 L of sterile growth medium (pH 8.1 ± 0.1) and an acetone washed, sterilised star shaped stirring bar were added to the bottle to achieve a minimum headspace.
The solution was stirred slowly avoiding bubble and foam formation for 24 hours at room temperature (about 20°C) to achieve maximum solubility of the test item. Another flask was filled with growth medium only for the control replicates and stirred in the same way.

To separate insoluble test item from the test medium the bottles stood for 1 hour. Thereafter, 100 mL were drained off and discarded. The next slowly released 2 L were used for the testing and adjusted to a pH of 8.1 ± 0.1. The test solution was checked for a Tyndall effect using a light source. No microdroplets or micells were observed and therefore, the solution was not centrifuged and immediately used for the testing. The four lower test concentrations were prepared by serial dilution.

All work was conducted on a clean bench using sterile equipment.

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata, Chlorophycea, Chlorophyta.
Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, 37073 Göttingen.
Strain number: Catalog No 61.81 SAG.
Cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guide-line (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing a pre-culture was established in OECD growth medium to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

As defined by OECD medium (OECD 201, Annex 3)

Test temperature:

22 °C (+/- 0 °C)

pH:

See IUCLID section "Any other information on results incl. tables".

Dissolved oxygen:

n.a.

Salinity:

n.a.

Conductivity:

not reported

Nominal and measured concentrations:

Nominal concentrations:
5.66, 11.3, 22.6, 45.3 and 90.5 mg a.s./L

The measured concentrations at test initiation were within ±20% of the nominal concentration (80.6 to 90.7 % of nominal).
Afterwards, the measured concentrations remained stable within ±20% of the nominal concentration (82.9 to 97.9 % of nominal).

Therefore, the evaluation of biological results was based on nominal concentrations as recommend in OECD guideline 201.

Details on test conditions:

Prior to the definitive test, a non-GLP range-finder to determine the maximum solubility of the test item in growth medium and the stability of the test item over 72 hours was conducted. Test concentrations of 9.07, 45.3 and 90.5 mg a.s./L were prepared comparable to the WAF approach as described in the OECD 23 as single WAFs to achieve a maximum solubility of the test item. Test vessels with “standard” and with “minimized” headspace (airtightly closed vessels) and with and without algae were prepared to determine if the test item will be degraded or volatilize over time.
At test start, 3.07 mg/L and 43.7 mg a.s./L were measured in the treatments of 9.07 and 90.5 mg a.s./L, respectively, corresponding to a maximum solubility of 34% to 48%. Afterwards, the test item remained stable over time regardless of whether the test was performed with headspace or minimized headspace in airtightly closed vessels. At the lower concentration of 9.07 mg/L (nominal) the increased presence of algae lead to a moderate decrease of the test item concentration after 72 hours.
At test termination (72 hours), the growth rate was affected by 0.9%, 56.1% and 56.6% at test concentrations of 9.07, 45.3 and 90.5 mg a.s./L (normal vessels). In the normal vessels (headspace, air-permeable closure) the pH of the test solution remained stable at around 8 over 72 hours, presumably due to the increased NaHCO3 concentration. However, in the vessels with minimal headspace closed airtightly, pH shifted to the alkaline in control and test vessels with nominal 9.07 mg/L test item (pH ca. 9.7).
Based on the results of the range-finder, a standard test design (headspace, air-permeable closure) was selected to provide for a constant pH during the test.
For further details, see IUCLID section "Any other information on materials and methods incl. tables".

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol, checked twice a year.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

measured concentrations within +/- 20% of nominal throughout the test

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 17.7-36.0

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.66 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

measured concentrations within +/- 20% of nominal throughout the test

Basis for effect:

growth rate

Remarks:

and Yield (biomass)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

measured concentrations within +/- 20% of nominal throughout the test

Basis for effect:

biomass

Remarks on result:

other: 95% C.I.: 6.49-11.24

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.22 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

measured concentrations within +/- 20% of nominal throughout the test

Basis for effect:

biomass

Remarks on result:

other: 95% C.I.: 4.83-8.02

Details on results:

Light intensity ranged from 95.31 to 101.37 µE m-2 s-1 and the temperature remained stable at 22.0 °C during the test.
The pH of the controls decreased during the test from 8.04 at test start to a mean value (n = 8) of 7.90 at test end. In the test concentrations, the initial pH in the treatments ranged from 8.08 to 8.13 and the mean pH was between 7.82 and 7.83 at test termination.
Further details on results are given in IUCLID section "Any other information on results incl. tables".

Results with reference substance (positive control):

3,5-dichlorophenol, results from March 2020: ErC50 = 2.92 mg/L (nominal; 95% confidence limits: 2.64 – 3.24 mg/L), which was in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.

Reported statistics and error estimates:

Biological data were statistically analysed to determine EC50 and EC10 values together with 95 % confidence intervals, where possible.
The EyC10 and EyC50 were determined using the 3-parametric non-linear regression procedure. There was a lack of fit for the tested non-linear regression models (3-parametric normal, logistic and Weibull) and therefore, the ErC50 was calculated based on linear regression procedures (Probit). No ErC10 could be calculated for growth rate, due to the steep increase of the curve of the sigmoid function caused by the strong inhibition beginning already at the second test concentration. Different linear (Probit, Logit, Weibull) and non-linear (3-parametric and 4-parametric normal, logistic and Weibull) regression models were used trying to calculate a reliable ErC10 value. However, either the regression model was found to be unsuitable (lack of fit) or no ErC10 could be calculated.

Due to normal distribution (p > 0.01) and variance homogeneity (Levene test, p > 0.01) the NOEC for growth rate was determined by the Williams` Multiple Sequential t-Test, whereas the NOEC for yield (Normality check failed, p <= 0.01; Levene test indicated variance heterogeneity, p <= 0.010) was determined by the Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (significance level of 0.05, one-sided smaller).

The computer program ToxRat® [1] was used for statistical evaluations.

[1] ToxRat® Professional 3.3.0. ToxRat® Solutions GmbH, Naheweg 15, 52477 Alsdorf, Germany.

Analytical results

The measured concentrations at test initiation were within 80 – 120% of nominal. Afterwards, the measured concentrations remained stable within ±20% of the measured initial as well as nominal concentrations throughout the test period of 72 hours. Therefore, the evaluation of biological results was based on nominal concentrations as recommend in OECD guideline 201. Details are given in the table below.

Table 1: Results of the chemical analysis of the test item at test initiation (0 h), after 24 and 48 hours and test termination (72 h):

Sampling date	Nominal conc.	Measured conc.	% of Nominal	% of initial
	[mg/L]	[mg/L]	[%]	[%]
Test initiation	Control	<LOQ	-	-
	5.66	5.13	90.7	-
	11.3	9.77	86.5	-
	22.6	18.7	82.9	-
	45.3	36.4	80.6	-
	90.5	73.1	80.7	-
24 hours	Control	<LOQ	-	-
	5.66	4.85	85.6	94.4
	11.3	9.56	84.6	97.8
	22.6	19.1	84.6	102
	45.3	37.5	82.9	103
	90.5	78.1	86.3	107
48 hours	Control	<LOQ	-	-
	5.66	5.51	97.3	107
	11.3	10.3	91.0	105
	22.6	20.5	90.9	110
	45.3	40.5	89.7	111
	90.5	81.9	90.5	112
Test termination (72 hours)	Control	<LOQ	-	-
	5.66	5.53	97.8	108
	11.3	11.0	97.5	113
	22.6	22.1	97.9	118
	45.3	41.4	91.5	113
	90.5	86.8	95.9	119

Biological Results

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

Table 2: Inhibition of growth rate and yield compared to controls after 72 hours

Nominal conc. [mg a.s./L]	Growth rate		Yield
	Growth rate [1/d]	% Inhibition	Yield [cells x 10^4/mL]	% Inhibition
Control	1.401	0	33.3	0
5.66	1.386	1.1 (-)	31.7	4.7 (-)
11.3	0.729	48.0 (+)	4.0	88.1 (+)
22.6	0.589	57.9 (+)	2.4	92.7 (+)
45.3	0.564	59.8 (+)	2.2	93.3 (+)
90.5	0.460	67.1 (+)	1.5	95.5 (+)

(+) significantly/ (-) not significantly different from controls, Williams` Multiple Sequential t-Test (growth rate) and Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm (yield), significance level 0.05, one-sided smaller.

Table 3: Effective concentrations based on nominal concentrations of the test item for the exposure of R. subcapitata for 72 hours

Parameter		EC10	EC20	EC50	LOEC	NOEC
Growth rate	Value [mg a.s./L]	n.d.	5.15	25.0	11.3	5.66
	Lower 95 %-cl	n.d.	1.91	17.7	-	-
	Upper 95 %-cl	n.d.	8.52	36.0	-	-
Yield	Value [mg a.s./L]	6.22	6.93	8.50	11.3	5.66
	Lower 95 %-cl	4.83	5.48	6.49	-	-
	Upper 95 %-cl	8.02	8.81	11.24	-	-

n.d.: not determined due to inappropriate data or mathematical reasons.

Microscopic examinations were performed to verify a normal and healthy appearance of the inoculum culture and to detect any abnormal appearance of the algae (as may be caused by the exposure to the test substance) at the end of the test.

Table 4: Microscopic observation of the algae cells at test initiation and termination of the 72 h growth test

Date	Nominal conc. [mg a.s./L]	Observations
Test start	Normal appearance of inoculum culture for all test concentrations.
Test end, 72 h	Control	Normal appearance of algal cells.
	5.66	Normal appearance of algal cells.
	11.3	Significantly fewer algal cells than in the control.
	22.6	Significantly fewer algal cells than in the control.
	45.3	Appearance of only a few algal cells. Mainly cell debris.
	90.5	Appearance of only a few algal cells. Mainly cell debris.

Table 5: Cell numbers (x 10^4) per mL dependent on nominal concentrations and time

Nominal conc. [mg a.s./L]	Control	5.66	11.3	22.6	45.2	90.5
0 h	0.5	0.5	0.5	0.5	0.5	0.5
24 h	2.1	1.6	1.7	1.6	1.4	1.2
	1.8	1.7	1.5	1.6	1.3	1.4
	2.3	2.2	1.7	1.7	1.2	1.2
	1.8	1.8	1.7	1.5	1.3	1.4
	1.8
	1.6
	1.6
	2.1
Mean:	1.9	1.8	1.6	1.6	1.3	1.3
Std.Dev.:	0.2	0.3	0.1	0.1	0.1	0.1
n:	8	4	4	4	4	4
CV:	12.3	13.7	6.2	3.7	4.2	7.2
48 h	6.7	6.3	3.6	3.0	1.9	1.4
	6.2	6.2	3.3	2.4	1.9	1.1
	6.8	6.5	3.4	2.5	1.6	1.3
	6.9	6.8	3.7	2.3	2.1	1.3
	6.4
	6.3
	6.5
	6.8
Mean:	6.6	6.4	3.5	2.6	1.9	1.3
Std.Dev.:	0.3	0.3	0.2	0.3	0.2	0.1
n:	8	4	4	4	4	4
CV:	4.1	3.9	5.9	11.3	11.4	8.4
72 h	37.9	29.4	4.8	3.2	2.8	2.2
	38.5	38.5	4.8	3.0	2.5	2.2
	36.8	30.4	4.1	2.8	2.5	1.9
	29.9	30.5	4.2	2.7	3.0	1.7
	32.5
	32.2
	25.8
	36.4
Mean:	33.8	32.2	4.5	2.9	2.7	2.0
Std.Dev.:	4.5	4.2	0.4	0.2	0.2	0.2
n:	8	4	4	4	4	4
CV:	13.2	13.2	8.8	8.2	8.6	10.9

Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

Cell number at test start: 5 000 cells/mL

Table 6: The pH of test solutions at test start and test end

	Nominal conc. [mg a.s./L]
	Control	5.66	11.3	22.6	45.2	90.5
Test start	8.04	8.09	8.08	8.10	8.13	8.10
Test end	7.94	7.90	7.78	7.85	7.83	7.83
	7.98	7.81	7.81	7.79	7.80	7.82
	7.93	7.82	7.87	7.86	7.84	7.81
	7.89	7.80	7.86	7.84	7.85	7.83
	7.89
	7.89
	7.85
	7.87
Mean	7.90	7.83	7.83	7.83	7.83	7.82
Minimum	7.85	7.80	7.78	7.79	7.80	7.81
Maximun	7.98	7.90	7.87	7.86	7.85	7.83

Validity criteria fulfilled:

yes

Remarks:

Control cell number increased by a factor of 67.5 within 72 h; mean of the control replicate CV (%) of section-by-section specific growth rate of controls: 16.1 %; CV (%) of average specific growth rates over 72h for control replicates: 3.3 %

Conclusions:

In a valid study on freshwater algae growth inhibition performed compliant with GLP according to OECD 201, the following results were obtained for the test item n-nonanoic acid:
NOEC (Raphidocelis subcapitata; 72 h; growth rate & yield; nominal, anal. verified) = 5.66 mg/L;
ErC50 (Raphidocelis subcapitata; 72 h; growth rate; nominal, anal. verified) = 25 mg/L (95% C.I.: 17.7-36.0);
ECy10 (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 6.22 mg/L (95% C.I.: 4.83-8.02);
ECy50 (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 8.50 mg/L (95% C.I.: 6.49-11.24).
All validity criteria of the guideline were met.

Executive summary:

A study was performed compliant with GLP at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the toxicity of Pelargonic acid on the growth of the unicellular freshwater green alga Raphidocelis subcapitata.

Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations: 5.66, 11.3, 22.6, 45.3 and 90.5 mg a.s./L and a control (factor: 2).

The concentrations of the test item in the media were confirmed by GC-MS (LOQ = 1.00 mg a.s./L) measurements at test initiation, after 24 hours, 48 hours and test termination after 72 h.

The measured concentrations ranged from 80.6 – 90.7% of nominal concentrations at test initiation and were within 80 – 120% of nominal. Afterwards, the measured concentrations remained stable within ±20% of the measured initial as well as nominal concentrations throughout the test period of 72 hours. Therefore, the evaluation of biological results was based on nominal concentrations as recommend in OECD guideline 201.

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

No ErC10 could be determined for the endpoint growth rate inhibition due to the steep increase of the curve of the sigmoid function caused by the strong inhibition beginning already at the second test concentration. Different linear (Probit, Logit, Weibull) and non-linear (3-parametric and 4-parametric normal, logistic and Weibull) regression models were tried, but either the regression model was found to be unsuitable (lack of fit) or no ErC10 could be calculated. However, the determined NOEC must be very close to the ErC10 in this case.

The following (No)effect concentrations could be determined:

NOEC (Raphidocelis subcapitata; 72 h; growth rate & yield; nominal, anal. verified) = 5.66 mg/L;

ErC50 (Raphidocelis subcapitata; 72 h; growth rate; nominal, anal. verified) = 25 mg/L (95% C.I.: 17.7-36.0);

ECy10  (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 6.22 mg/L (95% C.I.: 4.83-8.02);

ECy50  (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 8.50 mg/L (95% C.I.: 6.49-11.24).

All validity criteria of the guideline were met.

Description of key information

In a valid study on freshwater algae growth inhibition performed compliant with GLP according to OECD 201, the following results were obtained for the test item n-nonanoic acid:

NOEC (Raphidocelis subcapitata; 72 h; growth rate & yield; nominal, anal. verified) = 5.66 mg/L;

ErC50 (Raphidocelis subcapitata; 72 h; growth rate; nominal, anal. verified) = 25 mg/L (95% C.I.: 17.7-36.0);

ECy10  (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 6.22 mg/L (95% C.I.: 4.83-8.02);

ECy50  (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 8.50 mg/L (95% C.I.: 6.49-11.24).

All validity criteria of the guideline were met.

Key value for chemical safety assessment

EC50 for freshwater algae:

25 mg/L

EC10 or NOEC for freshwater algae:

5.66 mg/L

Additional information

A study was performed compliant with GLP at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the toxicity of Pelargonic acid on the growth of the unicellular freshwater green alga Raphidocelis subcapitata. Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations: 5.66, 11.3, 22.6, 45.3 and 90.5 mg a.s./L and a control (factor: 2).

The concentrations of the test item in the media were confirmed by GC-MS (LOQ = 1.00 mg a.s./L) measurements at test initiation, after 24 hours, 48 hours and test termination after 72 h.

The measured concentrations ranged from 80.6 – 90.7% of nominal concentrations at test initiation and were within 80 – 120% of nominal. Afterwards, the measured concentrations remained stable within ±20% of the measured initial as well as nominal concentrations throughout the test period of 72 hours. Therefore, the evaluation of biological results was based on nominal concentrations as recommend in OECD guideline 201.

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

No ErC10 could be determined for the endpoint growth rate inhibition due to the steep increase of the curve of the sigmoid function caused by the strong inhibition beginning already at the second test concentration. Different linear (Probit, Logit, Weibull) and non-linear (3-parametric and 4-parametric normal, logistic and Weibull) regression models were tried, but either the regression model was found to be unsuitable (lack of fit) or no ErC10 could be calculated. However, the determined NOEC must be very close to the ErC10 in this case.

The following (No)effect concentrations could be determined:

NOEC (Raphidocelis subcapitata; 72 h; growth rate & yield; nominal, anal. verified) = 5.66 mg/L;

ErC50 (Raphidocelis subcapitata; 72 h; growth rate; nominal, anal. verified) = 25 mg/L (95% C.I.: 17.7-36.0);

ECy10  (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 6.22 mg/L (95% C.I.: 4.83-8.02);

ECy50  (Raphidocelis subcapitata; 72 h; yield; nominal, anal. verified) = 8.50 mg/L (95% C.I.: 6.49-11.24).

All validity criteria of the guideline were met.