Butyraldehyde

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-08-12 - 2022-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Butyraldehyde

EC Number:

204-646-6

EC Name:

Butyraldehyde

Cas Number:

123-72-8

Molecular formula:

C4H8O

IUPAC Name:

butanal

Test material form:

liquid: volatile

Details on test material:

No data

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of the test material used in the study report: Butyraldehyde

- Source (i.e. manufacturer or supplier) and lot/batch number of test material: test material was supplied by Perstorp AB (the study sponsor)

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid form

Sampling and analysis

Analytical monitoring:

yes

Remarks:

sample was collected at 0h, 24h, 48h and 72h on the same day of exposure. additional duplicate and triplicates were collected and stored at 4 °C.

Details on sampling:

- Concentrations: Nominal Concentrations of the tested item were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L where the The geometric mean measured test concentrations were determined to be 0.0080, 0.038, 0.095, 0.89, 2.3 and 60 mg/L.
- Sampling method: A small volume of the algal suspension was withdrawn from each test flask after 24, 48 and 72 hours for the measurement of algal biomass and will not be replaced.
- Sample storage conditions before analysis: Water samples were analyzed immediately on the same day of collection. Duplicate and triplicate samples were frozen for later analysis if deemed necessary.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically, and any abnormalities recorded. Samples were taken at 22, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): 3 to 4 days
- Method of cultivation: inoculum cultures of algae was set up at 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 105 to 106 cells/mL.

ACCLIMATION
- Acclimation period: 3-4 days, acclimatization condition was same as the test condition

- Culturing media and conditions (same as test or not): The culture medium used for the range-finding and definitive tests was the same as that used to maintain the stock culture except the media used in tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with conducting the test in an enclosed system (Herman et al 1990).

- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

constant illumination and shaking was maintained during the exposure period.

Test conditions

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

24 ± 1 °C.

pH:

7.5 ± 0.1

Salinity:

N/A

Nominal and measured concentrations:

Concentrations of the test item (nominal): 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Concentrations of the test item (measured): 0.0080, 0.038, 0.095, 0.89, 2.3 and 60 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks
- Type (delete if not applicable): closed and static system, flasks were sealed with ground glass stoppers and incubated (INFORS Multitron incubator)
- Material, size, headspace, fill volume: 250mL glass conical flasks
- Aeration: constantly shaken at approximately 150 rpm for 72 hours.

- Renewal rate of test solution (frequency/flow rate): No media renewal
-Initial concentration of cell density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.41 x 106 cells per mL
- Control end cells density: algal suspension (5.3 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.
- No. of vessels per concentration (replicates): 6 vessels
- No. of vessels per control (replicates): 3 vessels

GROWTH MEDIUM
- Standard medium used: yes. The medium for the range finding and the definitive study was prepared according to OECD 201 (2006 version). except the media used in tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with conducting the test in an enclosed system (Herman et al 1990).


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Culture medium different from test medium: No
- Intervals of water quality measurement: water quality was measured during 0h and 72h

OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod &- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. initial nominal cell density of 5.00 x 103 cells/mL.
- Chlorophyll measurement: No


TEST CONCENTRATIONS
- Spacing factor for test concentrations: for the definitive test, a factor of 3.125 was used. Nominal tested concentrations used, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

- Range finding study
- Test concentrations: nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 72 hours.
- Results used to determine the conditions for the definitive study: The result of the range-finding study indicated the test item was not very stable and the mean measured concentrations were very low compared to the nominal concentrations.

CULTURING APPARATUS
-Details on culturing apparatus used: glass apparatus was used for the culture

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.1 mg/L

95% CI:

0.77 - 1.3

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.89 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2 mg/L

95% CI:

1.6 - 2.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.3 mg/L

95% CI:

5.8 - 9.4

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The geometric mean measured test concentrations were calculated using the Excel geometric mean functionality as follows: GM𝑦 = 𝑛(√𝑦1𝑦2𝑦3 𝑦𝑛)
Where:
GM = geometric mean measured test concentration (mg/L)
n = total number of observations
Yx = measured concentration at each time point (mg/L) Yn = measured concentration at the end of the test (mg/L)
Where a measured test concentration of less than the LOQ or none detected was obtained, a value of half the LOQ was substituted into the equation

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities were observed
- Adherence to test vessels: the stability test during the range finder and the additional vessels added for each concentrations (without algae) during the definitive test showed no adherence to the glass beaker.
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the substance was most likely to be lost from the test system due to the high volatility

Inhibition of growth rate:
ErC10 (0 to 72 hour) : 1.1 mg/L; 95% confidence limits 0.77 to 1.3 mg/L
ErC20 (0 to 72 hour) : 2.0 mg/L; 95% confidence limits 1.6 to 2.5 mg/L
ErC50 (0 to 72 hour) : 7.3 mg/L; 95% confidence limits 5.8 to 9.4 mg/L
where ErCx is the test concentration that reduced growth rate by x%.

Inhibition of Yied:
EyC10 (0 to 72 hour) : 0.87 mg/L; 95% confidence limits 0.59 to 1.1 mg/L
EyC20 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 0.89 to 1.4 mg/L
EyC50 (0 to 72 hour) : 1.8 mg/L; 95% confidence limits 1.5 to 2.1 mg/L
Where EyCx is the test concentration that reduced yield by x%.

According to the OECD 201 TG, the growth rate was selected as the primary endpoint and the yield was considered as a secondary endpoint.

-The following observations were made to confirm the validity of the study.

1. Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL
Mean cell density of control at 72 hours : 6.34 x 105 cells per mL
2. The mean coefficient of variation for section by section specific growth rate for the control cultures was 15%
3. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 3%

Results with reference substance (positive control):

A positive control (Labcorp study number 8510841) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 12 December 2022 and 15 December 2022.

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.1 mg/L; 95% confidence limits 1.0 to 1.1 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.40 to 0.48 mg/L
No Observed Effect Concentration based on growth rate: 0.125 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

Reported statistics and error estimates:

Prior to determination of the Lowest Observed Effect Concentration (LOEC) and No
Observed Effect Concentration (NOEC), data for each endpoint was assessed using
Shapiro-Wilk’s test on Normal Distribution, Levene’s test on Variance Homogeneity and
Trend analysis by contrasts to enable the correct comparison test to be performed. The
Williams Multiple Sequential t-test Procedure was used for both growth rate and yield.
All statistical analyses were performed using the ToxRatPro software package.

Any other information on results incl. tables

The following computerized systems were used in the study:
Coulter® Multisizer 3 Particle Counter (3.53)
Cell density determinations
ToxRat (Version 3.3.0)
Professional computer software package
Veeva – Unified Quality Vault
Quality Management and Document Control System

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

For the static algae growth inhibition test conducted with Raphidocelis subcapitata applying nominal concentrations of 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal) where the measured concentrations were 0.0080, 0.038, 0.095, 0.89, 2.3 and 60 mg/L of Butyraldehyde, results based on growth rate after 72 h were as follows:
EC50: 7.3 mg/L (95 % C.I. 5.8 – 9.4 mg/L);
EC20: 2.0 mg/L (95 % C.I. 1.6 – 2.5 mg/L);
EC10: 1.1 mg/L (95 % C.I 0.77 – 1.3 mg/L)
LOEC: 2.3 mg/L;
NOEC: 0.89 mg/L.
As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.
All the following validity criteria were met,
The cell concentration of the control cultures must increase by a factor of at least 16 over the test period
• The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3, for the 72-Hour tests) must not exceed 35%
• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%

Executive summary:

In a 72 hour acute toxicity study, the cultures of Raphidocelis subcapitata were exposed to n-butyraldehyde at nominal concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L/ (measured concentrations of 0.0080, 0.038, 0.095, 0.89, 2.3 and 60 mg/L. respectively) under static or renewal conditions in accordance with the OECD 201 (2006 version).  The NOECr and ErC₅₀ values based on cell density were 0.89 mg/L and 7.3 [5.8 to 9.4] mg/L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from 3% to 100%.

 

The following abnormalities were noted:  (list abnormalities that may include unusual cell shape, colour differences, flocculation, adherence of algae to test vessels, aggregation of algal cell, precipitation in the test solution, or stimulation).  [If toxicity and abnormalities were not observed, state “There were no compound related phytotoxic effects.”]

Results Synopsis

 

Test Organism: Raphidocelis subcapitata

Test Type (Flowthrough, Static, Static Renewal):

 

72hr ErC₅₀:  7.3 mg/L                95% C.I.:  {5.8 to 9.4 mg/L}

72hr ErC₁₀:  1.1 mg/L                95% C.I.:  {0.77 to 1.3  mg/L}

72hr NOEC growth rate:  0.89     

Endpoint(s) Effected:  growth rate