Carboxylic acids, C5-9

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours 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:

For sample preparation the water-accommodated fractions for every treatments were prepared for the test. This was done by mixing the test item with the corresponding amount of nutrient medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 hours. The mixing period was validated by the following preliminary investigational work:
A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for chemical analysis. Whilst the results suggest that near nominal concentrations were obtained from the 24-Hour preparation, visual inspection of the WAF at the end of the settlement period indicated that the majority of the test item added remained at the water surface. As such it was considered likely that the analysis was detecting only relatively water soluble component of the test item, the measured concentrations provided not being indicative of the test item as a whole. As a slight decline in the measured concentration obtained was observed between the 24 and 96-Hour preparations, therefore, for the purpose of testing the test item was prepared as a WAF using a 23-Hour stirring period followed by a 1-Hour settlement period prior to removal of the aqueous phase for testing.

Test organisms (species):

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

Details on test organisms:

Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 104 to 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Culture Medium
NaNO3 25.5mg/L
MgCl2.6H2O 12.16mg/L
CaCl2.2H2O 4.41mg/L
MgSO4.7H2O 14.6mg/L
K2HPO4 1.044mg/L
NaHCO3 15.0mg/L
H3BO3 0.186mg/L
MnCl2.4H2O 0.415mg/L
ZnCl2 0.00327mg/L
FeCl3.6H2O 0.160mg/L
CoCl2.6H2O 0.00143mg/L
Na2MoO4.2H2O 0.00726mg/L
CuCl2.2H2O 0.000012mg/L
Na2EDTA.2H2O 0.30mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Test temperature:

24+/-1

pH:

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
A concentration dependent decline in pH was observed in the test preparation at 0 hours in the range of pH 7.6 at 1.0 mg/L loading rate WAF through to pH 4.5 at 100 mg/L loading rate WAF. This was considered to be due to an intrinsic property of the test item and as such no adjustment was made to the pH of the test preparations prior to the addition of algal cells at the start of the test.

Nominal and measured concentrations:

Based on the preliminary test loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of 6.6 and 81 mg/L respectively were obtained. A significant decline in measured concentration was observed in the 10 mg/L loading rate WAF at 72 hours to a level which could not be quantified. A measured concentration of 75 mg/L was obtained from the 100 mg/L loading rate WAF test preparation. Such an effect was considered to be due to adsorption of the test item to the algal cells present, there being greater numbers of algal cells in the lower concentration and hence greater surface area for adsorption to occur.

Details on test conditions:

Nominal amounts of test item (20, 32, 20, 64 and 200 mg) were each separately added to the surface of 20, 10, 2.0, 2.0 and 2.0 liters of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.2 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as a positive control

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

No Observed Effect Loading Rate

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

loading rate WAF

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

No Observed Effect Loading Rate

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading Rate

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOELR

Remarks:

Lowest Observed Effect Loading Rate

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Remarks:

Lowest Observed Effect Loading Rate

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

cell number

Details on results:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.77 to 85 mg/L. A significant, concentration dependent decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.023 mg/L, to 91 mg/L suggesting that the test item was adsorbing to the algal cells present.
Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L No Observed Effect Concentration based on yield: 0.25 mg/L Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate 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) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates 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).

 Cell Densities (cells x mL)

The cell densities were determined by photometric measurement of optical density. Cell numbers of individual replicates are given. The means aof the cell numbers of the control and the treatments are presented in the following table together the pH values:

Nominal Concentration in mg/L	Parameter	Cell Densities
		0 h	26 h	46 h	72 h
0	Mean		31100	129000	789000
0	pH	7.7			8.8
1.0	Mean		34700	149000	997000
1.0	pH	7.6			8.9
3.2	Mean		34800	137000	949000
3.2	pH	7.5			8.9
10	Mean		34700	149000	997000
10	pH	7.4			8.4
32	Mean		8610	13900	41200
32	pH	6.2			7.9
100	Mean		5150	4870	7190
100	pH	4.5			4.7

Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Inhibition

The following mean inhibition values were calculated for the Growth Rate and the Yield.

Nominal loading rate in mg/L			Growth rate (cells/ml/hour)	% Inhibition	Yield (cells/mL)	% Inhibition*
0		Mean	0.070		784000
		SD	0.003		140000
1.0		Mean	0.074	[5]	992000	[27]
		SD	0.001		66600
3.2		Mean	0.073	[4]	944000	[20]
		SD	0.002		123000
10		Mean	0.070	0	778000	1
		SD	0.000		7180
32		Mean	0.029	59	36200	95
		SD	0.004		11800
100		Mean	0.003	95	1300	100
		SD	0.002		810

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated.

SD = Standard Deviation

[ ] = Increase in growth compared to controls

Validity criteria fulfilled:

yes

Remarks:

The results of the test are considered valid because all the fixed criteria of the guideline were met

Conclusions:

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.77 to 85 mg/L. A significant, concentration dependent decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.023 mg/L, to 91 mg/L suggesting that the test item was adsorbing to the algal cells present.
Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response EL50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect (mg/L) Lowest Observed Effect (mg/l)
Variable Loading Rate WAF Loading Rate WAF Loading Rate (NOEL) Loading Rate (LOEL)

Growth Rate 29 26 - 33 10 32
Yield 16 Not determined due to nature of data 10 32

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Test performed in 2001.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study following OECD 201 guideline and performed by Mitsubishi for the Japanese governement. Deviations: 3 replicates for the control group instead of 6 and no monitoring of oxygen concentration

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

3 replicates for the control group instead of 6 ; no monitoring of oxygen concentration.

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): n-Heptanoic acid
- Substance type: acid
- Physical state: Liquid
- Analytical purity: >97.7%
- Lot/batch No.: FHH02

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control (0 mg/L) ; 10 ; 18 ; 32.1 ; 56 and 100 mg/L
- Sampling method: tank water from each tank was analysed at 0 h (fresh media) and 24 h(expired media)
- Sample storage conditions before analysis: not indicated, but probably assayed right after sampling
Test solutions

Vehicle:

no

Details on test solutions:

OECD Medium according to the OECD 201 guideline

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Management after Obtaining: Successive aseptic culturing using Gorham agar
- Before Culturing: Pre-culturing period: 30 August 2002 - 3 September 2002
- During this period, the algae proliferated logarithmically. (The environmental conditions were the same as the test)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

from 23.4 to 24.2 °C

pH:

from 5.7 to 9.8

Dissolved oxygen:

Not specified

Salinity:

Not added

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal: 10 ; 15 ; 22 ; 32 ; 46 ; 68 and 100 mg/L
Measured at 0h: 10.7 ; 14.7 ; 21.1 ; 31 ; 48.4 ; 70.4 and 108 mg/L
Measured at 72h: 7.9 ; 13.5 ; 19.2 ; 27.5 ; 39.9 ; 62.6 and 86.6 mg/L

Since measured concentration did not vary more than 20% of the nominal concentrations, nominal concentrations were considered for EC50 and NOEC calculations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass conical flasks
- Type (delete if not applicable): closed with breathable silicone plugs
- Static conditions
- Initial cells density: 10 000 cells/mL
- Control end cells density:2.44 millions of cell per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (according to OECD 201 guideline)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: 4000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Particle couting equipment + Optical microscope

TEST CONCENTRATIONS
- Test concentrations: 0 ; 10 ; 15 ; 22 ; 32 ; 46 ; 68 and 100 mg/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

0-72

Effect conc.:

52.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

0-72

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

24-48

Effect conc.:

55.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

CL95=40.1-77.2 mg/l

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

24-48

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

24-72

Effect conc.:

56.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

CL95=36.9-86.4

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

24-72

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

Not applicable

Table 1: Heptanoic acid concentrations at the beginning and at end of the test

	Measured concentration (mg/l)
Nominal concentrations (mg/l)	0 h	% nominal	72 h	% nominal
Control	<0.1	-	<0.1	-
10	10.7	107	7.9	79
15	14.7	98	13.5	90
22	21.1	96	19.2	87
32	31	97	27.5	86
46	48.4	105	39.9	87
68	70.4	104	62.6	92
100	108	108	86.6	87

Table 2: pH monitoring during the test

	pH
Nominal concentrations (mg/l)	0 h	72 h
Control	7.7	9.7
10	7.6	9.7
15	7.2	9.8
22	7.1	9.8
32	7.0	9.6
46	6.8	8.6
68	6.5	7.8
100	5.7	5.6

Validity criteria fulfilled:

yes

Conclusions:

Pseudokirchnerella subcapitata: Heptanoic acid (CAS 114-14-8): 72-h NOEC: 29 mg/L, 72-h ErC50 = 60 mg/L (NITE, 2002)

Executive summary:

The growth inhibiting effect of heptanoic acid (CAS 114-14-8) on algae was tested withPseudokirchnerella subcapitata. The exposure duration was 72 h. The effect values were: 72 -h NOEC: 29 mg/L, 72 -h EC50: 60 mg/L (NITE, 2002). All validity criteria were fulfilled.

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response          EL50 (mg/L)          95% Confidence Limits (mg/L)               No Observed Effect (mg/L)          Lowest Observed Effect (mg/l)

Variable       Loading Rate WAF     Loading Rate WAF                                 Loading Rate (NOEL)                   Loading Rate (LOEL)

Growth Rate           29                                 26  -  33                                                   10                                                     32

Yield                       16               Not determined due to nature of data                         10                                                     32

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Key value for chemical safety assessment

EC50 for freshwater algae:

16 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.77 to 85 mg/L. A significant, concentration dependent decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.023 mg/L, to 91 mg/L suggesting that the test item was adsorbing to the algal cells present.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Other data from the constituent Heptanoic acid were also used to cover this endpoint.

The growth inhibiting effect of heptanoic acid (CAS 114-14-8) on algae was tested withPseudokirchnerella subcapitata. The exposure duration was 72 h. The effect values were: 72 -h NOEC: 29 mg/L, 72 -h EC50: 60 mg/L (NITE, 2009).