Reaction mass of 1,3-diisopropylbenzene and 1,4-diisopropylbenzene

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:

weight of evidence

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECD Guidance document on Aquatic Toxicity of Difficult Substances and Mixtures, 2000

GLP compliance:

yes

Specific details on test material used for the study:

DEB Blend prepared from equal volumes of diethylbenzene samples provided by Sterling Chemicals, Inc., BP Amoco Chemical Company, and The Dow Chemical Company.

Source: The diethylbenzene blend was comprised of equal volumes from three diethylbenzene samples. The first diethylbenzene sample was received from Sterling Chemicals Inc. on December 16, 2002, and was assigned ABC reference number TS-15053. The second and third diethylbenzene samples were received on December 20,2002, from BP Amoco Chemical Company and Dow Chemical, and were assigned ABC reference number TS-15 100 and TS-15 101, respectively.

Prep: The diethylbenzene blend was prepared on January 28,2003, by combining 410-mL volumes from each diethylbemene sample into a 2-L glass carboy. The carboy was capped, shaken by hand, and then placed on an orbital shaker for one hour. The mixed contents in the carboy, diethylbenzene blend, were transferred to two appropriately labeled 32-once amber bottles that were assigned ABC reference numbers
TS-15139 and TS-15140.

ABC reference number TS-15140 was used to prepare matrix spiking solutions and ABC reference number TS-15139 was used to prepare exposure solutions during the range-finding and definitive tests.

Purity: 92.3% and the sample was assigned an expiration date of February 11,2004, by ABC Laboratories, Inc

Analytical monitoring:

yes

Details on sampling:

The concentration of the diethylbenzene blend was determined in test solution samples collected at 0, 24,48 and 72 hours of the definitive test. Diethylbenzene blend-fortified samples were also preparedand analyzed at each sample period. The analysis of the test solution samples for diethylbenzeneblend was based on an analytical method validated in FWAM prior to the start of the definitive test. The validation was conducted under ABC Laboratories' Study No. 47927 (4). Results of the validation demonstrated that the analytical method was suitable for recovery of the diethylbenzene
blend in FWAM.

At 0 hour, a 45-mL volume was collected from the parent "new" solutions of the control, vehicle control, and each test substance treatment. At 24,48, and 72 hours, a 45-mL composite sample was collected from the three replicates of the contril and each treatment solution and was comprised of
15-mL volumes removed fiom each replicate. Quality control (QC) samples were prepared by fortifying control dilution water with the diethylbenzene blend at concentrations of 0.298 and 8.96 mg a.i./L. All samples were extracted three times with 5 mL of toluene per extraction. After each extraction, the organic and aqueous phases were allowed to separate and the upper, organic phase was removed and transferred to a pre-calibrated culture tube. The toluene was then evaporated under a gentle stream of nitrogen gas to the appropriate volume for analysis. The final concentration of analyte was within the range of the analytical reference standards (3.16 to 190 mg a.i./L) used to generate the calibration curve.

Vehicle:

yes

Details on test solutions:

Individual test solutions were prepared by adding an appropriate volume of a working standard or primary standard to 2.0 L of freshwater algae media resulting in nominal concentrations of 0.50, 1.0, 2.0, 4.0 and 8.0 mg a.i./L. Working standards were prepared in acetone. The vehicle control contained an acetone concentration of 0.10 mL/L. Each 2.0-L volume of the controls and test substance treatments was covered with a glass plate stirred and with a Teflon stir bar for approximately one hour. The stirring was adjusted to provide a vortex <10% of the solution depth. After stirring, the phases were allowed to separate for approximately 30 minutes. Beginning with the control and continuing up to the highest treatment, the bottom aqueous phase was drawn from the bottom of each bottle through the outlet into the test flasks. Flasks were not re-sampled because the test design called for zero headspace.

Freshwater Algal Nutrient Medium
The test medium was freshwater algal nutrient medium (FWAM) with 500 mg NaHCO,/L. The medium was prepared by the addition of appropriate reagent grade nutrients to ABC reagent water. After preparation, the algal medium was pH-adjusted to 7.5+/- 0.1 (using 0.1 N HC1) and filtered through a 0.45um Millipore filter. The 500 mg NaHCO3/L was added to the algal medium following filtration.

Test organisms (species):

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

Details on test organisms:

Initial algal cell concentrations were approximately 10000 cells/mL.

The parent stock of Selenastrum capricornutum used in the definitive test was obtained from the Department of Botany, Culture Collection ofAlgae, University of Texas at Austin, Austin, Texas, on January 22,2003. Periodically, new Selenmtrum capricornutum cultures were initiated using a portion of this parent stock or cloned from an existing culture derived from the parent stock. The algal culture used to inoculate this toxicity test was four days old at study initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

23.6-24.8°C

pH:

7.75-9.63 (Increased pH values were associated with algal growth)

Dissolved oxygen:

No data

Nominal and measured concentrations:

Nominal concentrations (mg a.i./L): 0 (control), 0 (vehicle control; 0.10 mL acetone/L), 0.50, 1.0, 2.0, 4.0, and 8.0 mg a.i./L
Measured concentrations (mg a.i./L): <0.0702 (control), <0.0702 (vehicle control), 0.292, 0.547, 1.14, 2.27, and 3.35 mg a.i./L

Details on test conditions:

Definitive Test
Test details: Static: nine replicates prepared at test initiation of which 3 replicates were sacrificed at each observation time point (24, 48, and 72 hours). Initial algal cell concentrations were approximately 10000 cells/mL. Test chambers were 125-mL Erlenmeyer flasks completely filled with test solution and sealed with a glass stopper. The volume in each flask was 145 mL. Flasks were incubated at 24 +/-2 C and with continuous oscillation of 100 rpm, under continuous illumination of 8,600 +/- 10% lux, provided by cool-white fluorescent lamps.

Range-Finding Test
A range-finding test was initiated on February 24, 2003, and utilized test concentrations of 0 (control), 0.10, 1.0, 10, and 20 mg a.i./L. The 10 and 20-mg a.i./L treatments were individually prepared and each was stirred for 30 minutes prior to use. The 0.10, and 1.0-mg a.i./L treatments were prepared by diluting an appropriate volume of the 10-mg a.i./L treatment. Immediately following preparation, an oily-like film was observed on the surface of the 20-mg a.i./L treatment. The control and each test concentration were replicated two times. After 72 hours of incubation, percent inhibition in cell growth as compared to the control was 0, 0, 0, 99, and 99% at nominal concentrations of 0.10,1.0,10, and 20 mg a.i./L, respectively. These results were used to establish the definitive test concentrations at 0 (control), 0 (vehicle control; 0.10 mI, acetoneL), 0.50,1.0,2.0,
4.0, and 8.0 mg a.i./L.

Definitive Test
The in-life phase of the definitive test was conducted ftom March 18 to 21,2003. The definitive test was performed using 125-mL Erlenmeyer flasks with glass stoppers. Prior to test initiation, the flasks were cleaned and autoclaved according to ABC standard operating procedures. The volume in each flask was approximately 145 mL with no headspace. Nine replicates were used for the control, vehicle control and each test concentration. A tenth replicate (abiotic control) of the lowest treatment level, 0.50 mg a.i./L, was also prepared, but was not inoculated with algae. This abiotic control was used to test for possible incorporation of the test substance into the algal biomass. All test solutions and the vehicle control contained equal solvent concentrations (0.1 ml acetone/L). The control contained no test substance or solvent. Each flask was labeled with the ABC study number, concentration, replicate, and grid position. The flasks were randomly positioned on a rotary shaker set at approximately 100 rpm and were incubated at 24 +/- 2C for 72 hours in a temperature controlled enclosure. Continuous cool-white fluorescent lighting was provided with a target light intensity of 8,600 +/- 10% lux. Environmental chamber temperature and light intensity were monitored throughout the study. The temperature of the environmental chamber was monitored continuously during the definitive test using an electronic data-logger. The light intensity was measured with a LI-COR Model LI-189 light meter equipped with a photometric sensor.

At test initiation, an 80-mg a.i./mL primary standard was prepared by diluting approximately 2.1670 g of the diethylbenzene blend (2.0001 g corrected for purity) with acetone to a 25-mL volume. Volumes of the 80-mg a.i./mL primary standard were diluted with acetone to prepare working standards at 5.0,10,20, and 40 mg a.i./mL. A 0.20-mL volume of the appropriate working standard and the primary standard was added to 2.0 L of FWAM in a 2.0-L glass aspirator bottle to prepare the test substance treatments. The vehicle control was prepared by adding 0.20 mI, of acetone to 2.0 L of FWAM. Each 2.0-L volume of the controls and test substance treatments was covered with a glass plate stirred with a teflon stir bar for approximately one hour. The stirring was adjusted to provide a vortex 40% of the solution depth. When stirring was terminated, the phases were allowed to separate for approximately 30 minutes. Beginning with the control and continuing up to the highest treatment, the bottom aqueous phase was drawn fiom the bottom of each bottle through the outlet into the test flasks. Each test flask was completely filled with solution and sealed with a glass stopper. After each test flask was filled, an additional volume was collected for analytical confirmation andlor water quality measurements. Prior to siphoning, the 8.0 mg/L test solution was clear with an oily surface film. All remaining solutions were clear with no visible precipitate or surface film.

The definitive test was conducted for 72 hours commencing when the test flasks were inoculated with algae. Each flask was inoculated with 1.0 mL of algae containing approximately 1.0 x 106 cells/mL, resulting in approximately 1.0 x 104 cells/mL for each flask. Cell counts were made using using a light microscope and a hemocytometer for one set of triplicate flasks of control, vehicle control, and each test substance treatment once every 24 hours. In addition to cell density determinations, microscopic examination was conducted to determine if there were any morphological or physical effects on the algal cells. Following the 24-hour and 48-hour cell counts, the triplicate sets were discarded.

At 0 hour, measurements of temperature and pH were made in samples of the control, vehicle control, and test substance treatment parent solutions. The 72-hour measurements were made in replicate I of the controls and all test substance treatments. Temperature and pH were measured using a Denver Instruments pH meter.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.21 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.- 0.650-1.78 mg a.i./L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I.- 0-2.81 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.547 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Method Validation Results
Recoveries of diethylbenzene blend in the samples ranged fiom 93 to 11 1% of the nominal concentrations. No residues of diethylbenzene blend were detected in the controls at or above the MQL of 0.0632 mg ai./L. The recoveries indicated that the method was adequate for recovering diethylbenzene blend from freshwater algal media.

Analytical Chemistry Results
Measured concentrations of the diethylbenzene blend in test solutions at 0 hour ranged from 0.337 to 3.55 mg a.i./L and from 44 to 67% of the nominal concentrations.The limited solubility of the diethylbenzene blend in the dilution water impacted the recovery of the diethylbenzene blend from 0-hour test solutions and the impact was the greatest in the highest test substance treatment. Measured concentrations of the diethylbenzene blend in test solutions at 24 hours ranged from 0.300 to 3.38 mg a.i./L and from 42 to 61% of the nominal concentrations. At 48 hours, the measured concentrations of the diethylbenzene blend in test solutions ranged from 0.325 to 3.54 mg a.i./L and from 44 to 65% of the nominal concentrations. Measured concentrations of the diethylbenzene blend in test solutions at 72 hours ranged from 0.207 to 2.93 mg a.i./L and from 37 to 56% of the nominal concentrations. No residues of the diethylbenzene blend were detected in the control or vehicle control solutions above the MQL of 0.0702 mg a.i./L. The recovery of the diethylbenzene blend from the abiotic test substance treatment at 72 hours was 55% of the nominal concentration, and was similar to the percent recovered from the equivalent biotic test substance treatment at the same time period. Recoveries of the diethylbenzene blend in the 80,000-mg a.i./L stock solution at 0 hour was 113% of the nominal concentration. QC spike recoveries ranged friom 92 to 111 % of the nominal concentrations with the exception of the 0-hour low QC spike where 64% of the diethylbenzene blend was recovered. The mean measured concentrations (mean of all measured concentrations) of the diethylbenzene blend ranged from 0.292 to 3.35 mg a.i./L and from 42 to 58% of the nominal concentrations. The mean measured concentrations were used to present the biological results.

No visible precipitate or surface film was observed in the test solutions throughout the definitive test. Following 48 hours of exposure, all treatments
Biological Results
Logarithmic phase growth was confirmed at 72 hours in the control with a mean cell density of 61 e4 cells/mL, which was a 61-fold increase over the initial target cell density. The control was evaluated for variability among replicates by calculating the coefficient of variation using 72-hour cell density numbers. The coefficient of variation, calculated as the standard deviation divided by the mean times 100, was 13%. The cell growth in the control over the 72-hour test period and the low variability in cell densities between control replicates demonstrated the acceptability of the test.
Algal cells observed during the exposure were normal in appearance. No unusual cell shapes, color differences, flocculation, adherence of algae to test chambers, or aggregation of algal cells were noted during the exposure.

Percent change in algal biomass, as measured by area under the growth curve, ranged from -4 to -100% after 72 hours of exposure to the diethylbenzene blend. One-way analysis of variance showed a significant reduction of biomass (p/=1.14 mg a.i as compared to the vehicle control at 72 hours. The 72-hour NOEC was 0.547 mg ai./L, based on the lack of a statistically significant reduction of biomass at this test concentration. Based on biomass, the 72-hour EbC50 was 1.00mg a.i./L .

Percent change in algal growth rate ranged from +2 to -108% after 72 hours of exposure to the diethylbenzene blend. One-way analysis of variance showed a significant reduction (p/= 1.14 mg a.i./L after 72 hours. The 72-hour NOEC was 0.547 mg a.i./L, based on the lack of a statistically significant reduction of growth rate at this test concentration. Based on growth rate, the 72-hour ErC50 was 1.21 mg a.i./L.

Water Quality
The temperature of the test solutions ranged from 23.6 to 24.0°C at test initiation and from 23.9 to 24.8°C at 72 hours. Data from the electronic data logger confirm that the temperature of the incubator remained between 23.5 and 24.0°C throughout the exposure period. The pH of the test and control solutions ranged from 7.75 to 7.92 at test initiation and from 7.76 to 9.63 at 72 hours. Increases in pH were associated with increased algal biomass.

Results with reference substance (positive control):

No data

Cell Density (x10000 cells/mL)

at:                   24h        48hr     72hr

Control             2.0        15        61

Vehicle Control 1.9        12        72

0.292 mg a.i./L 2.5        12        68

0.547 mg a.i./L  2.2        13        63

1.14 mg a.i./L    1.2        2.2       16

2.27 mg a.i./L    0.92      0.82     0.81

3.35                  1.0        0.71     0.77

Biomass (calculated as area under the growth curve) was statistically different for control and vehicle (solvent) control;

treatments were thus compared against vehicle control for biomass.

Growth rate was not statistically different for control and vehicle control; treatments were compared against pooled control values for growth.

Biomass and growth  rates of the 3 highest concentrations were statistically reduced at all  three observation times. The 72-hr NOEC for both biomass and growth was 0.547 mg a.i./L.

The temperature of the solutions ranged from 23.6 to 24.8 C and the pH  ranged from 7.75 to 9.63. Increased pH values were associated with algal  growth.

Remarks:  All results were based upon mean measured concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Based on growth rate, the 72 hour EC50 = 1. 21 mg a.i./L (95% confidence interval: 0.650 to 1.78 mg a.i./L) and biomass EC50 = 1.00 mg a.i./L (95% confidence interval: 0 to 2.81 mg a.i./L) for a representative freshwater unicellular green alga.
The 72-hour NOEC for biomass and growth inhibition was 0.547 mg a.i./L, based on the lack of a statistically significant reduction in either endpoint at this test concentration.

Executive summary:

A phytotoxicity test was conducted at ABC Laboratories, Inc., Columbia, Missouri, to determine the effect of a diethylbenzene blend on the green alga, Selenastrum capricornutum. The criterion for effect was reduction in growth. The definitive test was conducted for 72 hours commencing when the test flasks were inoculated with algae. Nine replicates were used for the control, vehicle control and each test concentration. A tenth replicate (abiotic control) of the lowest treatment level, 0.50 mg a.i./L, was also prepared, but was not inoculated with algae. Actual measured concentration of diethylbenzene (mg a.i./L): <0.0702 (control), <0.0702 (vehicle control),  0.292, 0.547, 1.14, 2.27, and 3.35 mg a.i./L.

Based on area under the growth curve, the 72-hour EbC50, for the diethylbenzene blend was 1.00 mg ai./L. The 72-hour NOEC, based on area under the growth curve, was 0.547 mg a.i./L. Based on growth rate, the 72-hour ErC50 for the diethylbenzene blend was 1.21 mg a.i./L. The 72-hour NOEC, based on growth rate, was 0.547 mg a.i/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Principles of method if other than guideline:

QSAR prediction using ECOSAR 1.11

GLP compliance:

no

Duration:

96 h

Dose descriptor:

EC50

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

other: growth reduction

Remarks on result:

other: No effect at saturation

Duration:

96 h

Dose descriptor:

other: ChV as calculated by ECOSAR

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

other: Growth reduction

Remarks on result:

other: No effect at saturation

Validity criteria fulfilled:

yes

Conclusions:

The isomers m-DIPB and p-DIPB were processed through ECOSAR 1.11 for algae toxicity prediction for the target substance DIPB mixture of isomers.
A QPRF was drafted in order to assess the 5 OECD principles regarding relevancy of the prediction.
The estimated algae EC50(96h) and ChV exceed by nearly a factor 10 the water solubilities of the two isomers. Therefore it is considered that the isomers and the target substances have no effect on algae at saturation.

Executive summary:

The isomers m-DIPB and p-DIPB were processed through ECOSAR 1.11 for algae toxicity prediction for the target substance DIPB mixture of isomers.

A QPRF was drafted in order to assess the 5 OECD principles regarding relevancy of the prediction.

The estimated algae EC50(96h) and ChV exceed by nearly a factor 10 the water solubilities of the two isomers. Therefore it is considered that the isomers and the target substances have no effect on algae at saturation. The criteria of acceptability (log Kow and molecular weight) are respected and the functional group of the target chemicals belongs to the application domain of “Neutral SAR”. Moreover there are an important number of chemicals in the training set for the Neutral SAR and the coefficient of determination related to the QSAR equation is good. Therefore the prediction can be estimated as relatively reliable, all the more that experimental data on analogues of the target chemicals present in the training set are in agreement with the predictions for the target chemicals. The predicted results are adequate for environmental classification and environmental risk assessment purpose.  

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

a) Stock Solution: 100 mg test substance was dissolved in 100 mg DMF with 300 mg HCO-40. The solution was diluted with OECD medium in 1000 mL.
b) Vehicle/Solvent and Concentrations: Dimethyl formamide(DMF) and HCO-40 were used for solvent. DMF 20.0 mg/L and HCO-40 60.0 mg/L were contained in each test solution and solvent control.

Test organisms (species):

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

Details on test organisms:

a) Supplier/Source: Obtained from American Type Culture Collection and reproduced in aseptic culture for 6 months.
b) Method of Cultivation: Sterile
c) Stain Number: ATCC22662

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Hardness:

OECD medium

Test temperature:

23+/-2 degrees C

pH:

OECD medium

Salinity:

Freshwater

Nominal and measured concentrations:

Nominal Concentrations: control, solvent control, 0.200, 0.430, 0.930, 2.00, 4.31, 9.28 20 mg/L

Details on test conditions:

a) Exposure Vessel Type: 100 mL Medium in an 500 mL Erlenmeyer Flask with glass cap (closed system)
b) Number of Replicates: 3
c) Initial Cell Number: 10,000 cells/mL
d) Water Temperature: 23+/-2 degrees C
e) Light Condition: 3,200 - 4,800 lux, continuously
f) Shaking: 100 rpm

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

a) Data Analysis: The EC50 values and associated 95% confidence limits were determined by least squares linear regression analysis of the logarithm of measured test concentration against percent growth inhibition relative to the solvent control. The NOEC values were determined by analysis of variance (ANOVA), Dunnet test, subsequent to Bartlett test for homogeneity of variances.
b) Method of Calculating Mean Measured Concentrations (i.e. arithmetic mean, geometric mean, etc.): Geometric mean of measured concentrations at 0 hr and at 72 hr were used for calculation.

Effect Data:
Area Method
EbC50(0-72hr) = 1.6 mg/L (mc) 
NOEC (0-72hr) = 0.31 mg/L (mc)

Measured Concentrations : 
The tested concentrations were measured at the start and the 72nd hour.   
The concentrations of 72 hours after were low. Those percent of nominal were 
17 - 23%. The test substance seemed to be incorporated into cell slightly.

---------------------------------------------------------
Nominal   Measured Conc., mg/L   Percent of nominal Mean*

conc.     --------------------------------------------
mg/L       0 hr      72 hr      0 hr    72 hr
---------------------------------------------------------
Control   <0.002    <0.002      ---     ---     ---
Solvent 
  Control <0.002    <0.002      ---     ---     ---
0.200      0.138     0.034      69      17      0.068
0.430      0.291     0.076      68      18      0.15
0.930      0.615     0.158      66      17      0.31
2.00       1.37      0.345      69      17      0.69
4.31       2.98      0.762      69      18      1.51
9.28       6.25      1.90       70      20      3.45
20.0       14.8      4.57       74      23      8.22
---------------------------------------------------------
* Geometric mean

Percent Growth Inhibition of Selenastrum capricornutum
---------------------------------------------------------
Measured          Area under the growth curves (Average)
Conc.             Area           Inhibition (%)*1
mg/L              A (0-72hr)      IA (0-72hr)
---------------------------------------------------------
Control             7,934,000         ---
Solvent Control    10,816,000         ---
0.068              10,995,000         -1.7
0.15               10,629,000         1.7
0.31                9,770,000         9.7
0.69                9,170,000        15.2*
1.51                6,651,000        38.5**
3.45                1,638,000        84.9**
8.22                 397,000         96.3**
---------------------------------------------------------

---------------------------------------------------------
                      Growth rates and percent
                       inhibition (Average)
Measured      -----------------------------------------
Conc.      Rate    Inhibition(%)*1  Rate Inhibition(%)*1
mg/L       u(24-48hr) Im(24-48hr)  u(24-72hr) Im(24-72hr)
---------------------------------------------------------
Control    0.0684       ---        0.0629      ---
Solvent 
 Control   0.0680       ---        0.0593      ---
0.068      0.0691      -1.6        0.0599      -1.0
0.15       0.0691      -1.6        0.0591       0.3
0.31       0.0661       2.8        0.0582       1.9
0.69       0.0648       4.7        0.0570       3.9
1.51       0.0552      18.8**      0.0526       11.3**
3.45       0.0300      55.9**      0.0227       61.7**
8.22       0.0094      86.2**      0.0057       90.4**
---------------------------------------------------------
*1 : Values are the percent inhibition relative to the control
*: Indicates a significant difference (alpha=0.05) from the solvent control
**: Indicates a significant difference (alpha=0.01) from the solvent  control

Water chemistry (pH) in test:
pH was measured for control and each concentration at the start and end of test.
pH: 8.1 - 10.

Calculation of toxicity values: The calculation of toxicity values was the mean measured concentrations. The reason is
that some of the error ranges of measured concentration were not less than 
+/-20% of nominal concentration.

Growth Curves: Log phase during the test period

NOEC  = 0.69 mg/L (growth rate), = 0.31 mg/L (biomass)
EC50  = 2.7 mg/L (growth rate),  = 1.6 mg/L (biomass)

Rate Method
ErC50(24-48hr) >= 3.05 mg/L (mc) (95% C.I.: 1.65-5.5 mg/L)
NOErC(24-48hr) = 0.69 mg/L (mc)
ErC50(24-72hr) >= 2.7 mg/L (mc)
NOErC = 0.69 mg/L (mc) 
mc: based on mean measured concentration

Validity criteria fulfilled:

yes

Conclusions:

In a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), Selenastrum capricornutum were exposed to diisopropylbenzene concentrations for 72 h in closed system and EC50 and NOEC in respect to biomass and growth rate determined. The test was conducted using solvent (mixture of a dispersant of HCO40 and DMF) and the estimated values were higher than water solubility, however, analytical monitoring was done. In respect to biomass the 72 h EbC50 and NOEC were determined to be 1.6 mg/L and 0.31 mg/L (measured concentrations) and in respect to growth rate the 72 h ErC50 and NOEC were found to be 2.7 mg/L and 0.69 mg/L (measured concentrations), respectively. However, all toxic signs were observed above the substance water solubility limits (0.0405 mg/l for p-DIPB and 0.072 mg/l for m-DIPB), so it can be concluded that there is no toxicity for test organisms up to water solubility limits.

Executive summary:

In a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), Selenastrum capricornutum were exposed to diisopropylbenzene concentrations for 72 h in closed system and EC50 and NOEC in respect to biomass and growth rate determined. The test was conducted using solvent (mixture of a dispersant of HCO40 and DMF) and the estimated values were higher than water solubility, however, analytical monitoring was done. In respect to biomass the 72 h EbC50 and NOEC were determined to be 1.6 mg/L and 0.31 mg/L (measured concentrations) and in respect to growth rate the 72 h ErC50 and NOEC were found to be 2.7 mg/L and 0.69 mg/L (measured concentrations), respectively.

However, all toxic signs were observed above the substance water solubility limits (0.0405 mg/l for p-DIPB and 0.072 mg/l for m-DIPB), so it can be concluded that there is no toxicity for test organisms up to water solubility limits.

Description of key information

In a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), Pseudokirchneriella subcapitata were exposed to diisopropylbenzene concentrations for 72 h in closed system. The test was conducted using solvent (mixture of a dispersant of HCO40 and DMF). Analytical monitoring was done. 72 h EbC50 and NOEC were determined to be 1.6 mg/L and 0.31 mg/L (measured concentrations) and 72 h ErC50 and NOEC were found to be 2.7 mg/L and 0.69 mg/L (measured concentrations), respectively. All these values were above the solubility limits of the compounds.

This was confirmed by the ECOSAR prediction. The isomers m-DIPB and p-DIPB were processed through ECOSAR 1.11 for algae toxicity prediction for the target substance DIPB mixture of isomers. A QPRF was drafted in order to assess the 5 OECD principles regarding relevancy of the prediction. The estimated algae EC50(96h) and ChV exceed by nearly a factor 10 the water solubilities of the two isomers. Therefore it is considered that the isomers and the target substances have no effect on algae at saturation. The criteria of acceptability (log Kow and molecular weight) are respected and the functional group of the target chemicals belongs to the application domain of “Neutral SAR”. Moreover there are an important number of chemicals in the training set for the Neutral SAR and the coefficient of determination related to the QSAR equation is good. Therefore the prediction can be estimated as relatively reliable, all the more that experimental data on analogues of the target chemicals present in the training set are in agreement with the predictions for the target chemicals.

The algae study performed on the analog Diethylbenzene blend also strengthen the fact that no effect is to be expected for algae at DIPB solubility limit (72-ErC50 and NOEC at 1.21 mg/L and 0.547 mg/L respectively).