3,5-dimethylbenzoyl chloride

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:

4 March 1996 - 14 March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Since DMBC rapidly hydrolyses in water, this study was effectively conducted on the hydrolysis product DMBA (dimethyl benzoic acid). All results are based on the mean measured concentrations of this compound, expressed as equivalent concentrations of DMBC.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethylbenzoyl chloride

EC Number:

413-010-9

EC Name:

3,5-dimethylbenzoyl chloride

Cas Number:

6613-44-1

Molecular formula:

C9H9ClO

IUPAC Name:

3,5-dimethylbenzoyl chloride

Details on test material:

DMBC
Appearance: colourless to yellow liquid
Storage conditions: in darkness at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Water samples (≈ 100 mL in duplicate) were taken from control and test cultures at 0 hours and at 72 hours (replicates pooled) and sent for analysis. The samples were not filtered to remove algal cells prior to analysis.

Additional water samples were also taken from flasks containing the test substance (highest exposure level) with no algae present, at 0 hours and at 72 hours (replicates pooled), in order to obtain information on the extent of adsorption of the test substance by the algal cells.

Test solutions

Vehicle:

no

Details on test solutions:

TEST SUBSTANCE PREPARATION
The test substance (200 mg) was initially dispersed in sterile nutrient medium (2 litres) with the use of ultrasonic disruption for approximately 10 minutes then stirred overnight with the aid of a magnetic stirrer to give an initial stock solution of 100 mg/L. Serial dilution of this stock solution with sterile nutrient medium produced the exposure series. A concentrated aliquot of algae was then introduced to each test level to obtain the desired starting density.

Prior to the addition of algae to the I00 mg/L exposure level, 500 mL was removed to obtain a 100 mg/L no algae test level. As the addition of the compound had a significant effect on the pH of the media, a second 500 mL sample was removed so that the pH could be adjusted to 7.6 with 1 M Sodium hydroxide solution.

TEST WATER
Sterile nutrient medium as detailed below.

CULTURE MEDIUM
Four stock solutions were prepared according to the following table, using reverse osmosis purified/deionised water. Stock solutions were sterilised by autoclaving (solutions 1-3) or by membrane filtration (solution 4) before being stored at +4 °C in the dark.

Aliquots of stock solutions 1 - 3 were further diluted with reverse osmosis purified/deionised water and autoclaved again to produce the working strength nutrient medium. Prior to use, an aliquot of stock solution 4 was added aseptically to the medium via a membrane filter. The pH of the medium after equilibration with air was approximately 8.

Nutrient Concentration Volume of stock solution Final concentration in
in stock solution per litre of final medium test solution

Stock solution 1: macro-nutrients
NH4Cl 1.5 g/L 10mL 15 mg/L
MgCl2.6H2O 1.2 g/L 12 mg/L
CaCl2.2H2O 1.8 g/L 18 mg/L
MgSO4.7H2O 1.5 g/L 15 mg/L
KH2PO4 0.16 g/L 1.6 mg/L


Stock solution 2: Fe-EDTA
FeCl3.6H2O 80 mg/L 1 mL 0.08 mg/L
Na2EDTA.2H2O 100 mg/L 0.1 mg/L


Stock solution 3: trace elements
H3BO3 185 mg/L 1 mL 0.185 mg/L
MnCl2.4H2O 415 mg/L 0.415 mg/L
ZnCl2 3 mg/L 3 x 10^-3 mg/L
CoCI2.6H2O 1.5 mg/L 1.5 x 10^-3 mg/L
CuCI2.2H2O 0.01 mg/L 10^-5 mg/L
Na2MoO4.2H2O 7 mg/L 7 x 10^-3 mg/L


Stock solution 4: NaHCO3
NaHCO3 50 g/L 1 mL 50 mg/L

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Centre of Algae & Protozoa c/o Freshwater Biological Association. Cumbria, UK.
-Pre-culture: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination 7000 lux) and stirring (orbital shaker) at 24 °C to give an algal suspension in log phase growth characterised by a cell density of 3.8 x 10^7 cells/mL.
The suspension was diluted using sterile nutrient medium to a cell density of 1.0 x 10^5 cells/mL prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 °C

pH:

As the compound had a severe impact on the pH of the test media, the top concentration was run in duplicate, with the pH adjusted to near neutral in one set of vessels.
At 100 mg/L nominal, the pH was found to be 3.9 - 4.0, due to the hydrolysis of DMBC to its acid. The vessel with pH adjusted to near neutral had a range of 7.5 - 9.8 over the 72 hours.

Nominal and measured concentrations:

Nominal test concentrations: 4.6, 10, 22, 46 and 100 (pH adjusted) mg DMBC equivalents/L.
Mean measured concentrations: 4.0, 8.8, 20, 42, 93 and 94 mg DMBC equivalents/L.

Details on test conditions:

EXPOSURE CONDITIONS
-Experimental design
Five test concentrations plus one untreated control, each in triplicate, were set up following preliminary range finding at 0.10 to 100 mg/L. The highest test concentration was also prepared with the pH adjusted with sodium hydroxide solution (1 M) to indicate if the toxic effects were pH mediated or as a direct toxic response to the test compound. Sub-culturing aliquots of the top concentration test solutions into fresh media after 72 hours demonstrated if the all cells were killed or if recovery was possible.

-Culture conditions
Conical flasks (250 mL) each containing 100 mL of test or control culture were covered with Nescofilm and placed at random in a Gallenkamp Illuminated Orbital Incubator. The cultures were incubated, without media renewal, for 72 hours under continuous illumination of approximately 7000 lux, provided by 7 x 30 W "universal white" 1 metre fluorescent tubes. Gaseous exchange and suspension of the algal cells was ensured by the action of the orbital shaker oscillating at 120 cycles per minute.

MEASUREMENT OF GROWTH
Samples were taken at 0, 24, 48 and 72 hours and the cell numbers were determined by direct counting with the aid of a haemocytometer (Improved Neubauer).

EVALUATION OF DATA
The area under each growth curve (cell density v time) is taken to be an index of growth.
Percentage inhibition of growth at each test concentration is calculated by comparing the area under the test curve with that under the control.

Percentage inhibition of growth values are plotted against test concentration, a line fitted by logistic regression and the EbC50 estimated by interpolation of the fitted curve. The EbC50 ("x" h) is the median effective concentration for inhibition of growth based on a comparison of areas under the growth curves after "x" hours.
The average specific growth rate for each exponentially growing culture is also calculated from the appropriate section of the growth curve.

Percentage reductions in growth rate and the ErC50 value are calculated as for the "area under the curve" data. The ErC50 ("x" - "y" h) is the median effective concentration for inhibition of growth based on a comparison of growth rates from "x" to "y" hours.
The "no-observed effect level" (NOEL) is obtained using Williams' test to compare the percentage inhibition in each treated group with that for the control cultures (Williams' D.A., 1971/72, biometrics 27; 103 - 117 and 28; 519 - 531).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 38 - 34 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1 - 78 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Details on results:

As DMBC was rapidly hydroylsed to 3,5-Dimethylbenzoyl acid (DMBA), in the conditions of the test, verification of the concentration of DMBA was carried out in fresh and expired media. However results were expressed as the arithmetic mean of the equivalent concentration of DMBC in fresh and expired media (Table 1). Measured concentrations ranged from 86 - 97 % of nominal at 0 hours and 90 - 98% of nominal at 72 hours.

Comparison of measured concentrations of the test substance obtained from water samples with and without algal cells present indicated that the presence of algal cells did not affect the stability of the exposure solutions.

The calculated "area under the curve" and "specific growth rate" values are given in Table 2 and are expressed in terms of percentage inhibition by comparing each value with that of the control curve. The models used to calculate EbC50 and ErC50 levels did not include data for the pH adjusted cultures.

Mean cell density of control at 0 h: 9.9 x 10^4 cells/mL.
Mean cell density of control at 72 h: 3.1 x 10^6 cells/mL.

At 100 mg/L nominal, the pH was found to be 3.9 - 4.0 and inhibition of the growth of the test species would normally be expected in these conditions. However in the pH adjusted cultures both the area under the curve and growth rate were inhibited compared to controls (63 % and 25 % respectively). This indicated that the growth inhibition observed was not purely pH mediated.

OBSERVATIONS
All test and control cultures were inspected microscopically at 72 hours. No abnormalities were detected in the control or any test cultures with the exception of the unadjusted 100 mg/L vessels. After 72 hours only cell debris was observed in these cultures.
No cultures showed any signs of contamination by foreign algal cells or protozoa.

Aliquots from the control and 100 mg/L test solutions (both pH adjusted and unadjusted) were sub-cultured to determine if the inhibitory effect of the test substance was algicidal or algistatic. Aliquots (10 mL) were removed from each replicate culture of the controls and test level and added to 90 mL of fresh sterile nutrient media to give a final volume of 100 mL. The cultures were recounted to provide an initial cell density then incubated for a further 72 hour period at 24 °C.

Regrowth occurred in all cultures and, consequently, the test substance is considered to be algistatic in effect.

Reported statistics and error estimates:

In the 93 mg/L group, no algal cells were found at 72 hours. Consequently, for the 0 - 72 hour growth rate data, the percentage inhibition of growth is infinite for this group which was therefore omitted from the analyses.

In order to estimate the concentration at which 50 % inhibition of growth occurs, logistic regression curves (against the logarithm of measured concentration) were fitted to the percentage inhibition values, excluding the pH adjusted group. For the 0 - 72 hour growth rate data, the upper asymptote of the fitted curve was fixed at the level of 100 % inhibition of growth. Although the fitted curves were not influenced by the control data, the confidence intervals for the 50 % point are adjusted for the control group variability.

Williams' test (Williams, 1971, 1972) was used to compare the percentage inhibition in each treated group with the baseline (control) values, excluding the pH adjusted group. The latter was compared separately with the control group using Student's I test. Bartlett's test for homogeneity of variance (Bartlett, 1937) was also applied.

For the 0 - 72 hour AUC data, Bartlett's test indicated significant heterogeneity of variance between the groups. For this data set, Williams' test was therefore repeated after excluding the 93 mg/L group. Since this did not result in any change to the conclusions drawn, the original analysis was retained.

DATA HANDLING
The data were entered by hand and analysed using Genstat 5 release 1.3 (Payne. et al, 1987).

Any other information on results incl. tables

Table 1 Measured Concentrations - Mean Values and Percentages of Nominal

Nominal Concentration mg/L	Number of Samples Analysed	Mean Measured Concentration mg/L*	% Nominal
Control 4.6 10 22 46 100 100**	2 2 2 2 2 2 2	Control 4.031 8.771 20.39 42.47 92.57 93.53	- 88 88 93 92 93 94

*DMBC equivalents as DMBA due to hydrolysis of DMBC

**pH adjusted test level

 

Table 2 Inhibition of growth

Nominal Concentration mg/L	Measured Concentration mg/L	Area Under the Curve at 72 hours	% Inhibition*	Growth Rate (0 - 72 h)	% Inhibition*
Control 4.6 10 22 46 100 100**	Control 4.0 8.8 20 42 93 94	7677 7628 7326 6451 1469 530 2866	- 1 5 16 81 107 63	0.0478 0.0477 0.0469 0.0442 0.0260 NC 0.0359	- 0 2 7 46 NC 25

*Percentage inhibition values calculated using non-rounded data

**pH adjusted test level

NC No calculation possible due to destruction of algal cells

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

DMBA, the hydrolysis product of DMBC, was inhibitory to the growth of algae at concentrations in excess of 8.8 mg DMBC equivalents/L. The EbC50 (72 h) is 31 mg DMBC equivalents/L and the ErC50 (0 - 72 h) is 44 mg DMBC equivalents/L.
The test substance is considered to be algistatic.

Executive summary:

A study was performed to assess the inhibitory effect of DMBC on the growth of the unicellular green alga Selenastrum capricornutum in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92169/EEC (O.J. No. L383A. 29.1192) Part C. Method 3 "Algal Inhibition Test" and the OECD Guideline for Testing of Chemicals No. 201 "Alga. Growth Inhibition Test".

 

Algal cultures were exposed to five test concentrations of DMBC (4.6, 10, 22. 46 and 100 mg/L nominal). As the compound had a severe impact on the pH of the test media, the top concentration was run in duplicate, with the pH adjusted to near neutral in one set of vessels to determine if the inhibition observed was due to the intrinsic toxicity of the compound or resulting from the acidic pH.

These test vessels, together with the untreated control, were incubated on an orbital shaker under continuous illumination at 24 °C for 72 hours. Growth was monitored daily by direct cell counting with the aid of an Improved Neubauer haemocytometer. At the end of the 72 hour exposure period, aliquots from the 100 mg/L exposure concentrations were sub-cultured into fresh media to determine if the compound was algicidal or algistatic.

 

The following values were derived from the data:

EbC50 72 h: 31 mg DMBC equivalents/L.

ErC50 0 - 72 h: 44 mg DMBC equivalents/L.

"No-observed effect level": 8.8 mg DMBC equivalents/L.

 

Measured concentrations ranged from 90 - 98 % of nominal at 72 hours. The results of the sub-culturing phase of the study indicated the compound was algistatic as the growth inhibition observed was not purely pH mediated.