4. STATISTICAL METHODS
4.2 Data Collection and Presentation [—
The North Carolina Pass/Fail Chronic Toxicity Test requires
a total of 10-days to complete even though the test oganisms are
only exposed to the control and test samples for 7-days. Day 1
is spent compositing the 24-hour effluent test sample and Day 2
is used to prepare and transport the test sample to the
analytical laboratory performing the test. On Day 3 the test is
initiated as the Ceriodaohnia are introduced to the control and
test samples. A single test organism is introduced into each of
twenty-four cups; of these, twelve are replicate controls
(comprised of culture/dilution water) and twelve are replicate
samples (comprised of the 24-hr composited effluent sample
diluted to the receiving instream waste concentration).
The remaining portion of the 24-hr effluent sample
composited on Day 1 is used to renew the test sample on Day 5,
whereas a second 24-hr effluent composite sample is collected on
Day 4 to renew the test sample on Day 8. Mortality and
reproduction counts are performed on Days 5, 8, and 10 . At the
-p-
FIGURE 4,1
STATISTICAL FLOW SCHEMATIC FOR ANALYZING
CERIODAPHNIA CHRONIC TOXICITY TEST DATA
Reproduction Data
Goodnoss of Fit or Shapiro-Wiil(
I
Passes Passes ?•I
Bartlstt's -^ Fails Dunnstt's T~3r
Passesl
Fails r^CHRONIC TEST FAILS
£1
Passes!
Fisher's Exact
CHRONIC TEST PASSES
Fails
Wilcoxon Rank Sum
I
Passes
J
3
Fails
10 11 1£ L L L 19 11 10 m 11 IZ L D D 15 15 14 surviving are counted and recorded, as well as the number of
neonates (juvenile Ceriodaphnia) borne per adult. Details of the
test are given in Section 2.3, pages 13 - 19.
Table 4.1 below illustrates a set of reproduction and mortality data from a typical pass/fail chronic toxicity test.
Table 4.1
Typical Set of Reproduction and Mortality Data
CONTROL SAMPLE
Replicate 1114^^289 Adult LLLLDLLLL Reproduction 20 12 18 17 9 15 8 17 20
TEST SAMPLE (97.6X Effluent)
Replicate 12.2.±k§.lS.l
Adult DLDDDDDDD Reproduction 11 7 6 12 13 17 10 10 11 where L = living and D = dead
The above set of data came from an actual test conducted by
Industrial & Environmental Analysts (Research Triangle Park, NC) on chlorinated effluent composited at the OWASA Mason Farm
Wastewater Treatment Plant (WWTP) during the first week of July, 1987. The first set of data corresponds to the control sample, whereas the second set corresponds to the test sample. The first
line (Replicate) refers to the number of the test vessel. Line two (Adult) refers to the living status of the adult
Ceriodaphnia. If an adult Ceriodaphnia expires within any time
during the test period (Day 3 through Day 10), a "D"
(representing dead) is placed under the respective replicate.
Should an adult Ceriodaphnia survive the duration of the test, an "L" (representing living) is placed under the respective
replicate. The third line (Reproduction) refers to the number of neonates borne to the adult Ceriodaohnia during the duration of the test.
Reproduction can occur even though the adult female
Ceriodaphnia may expire. This is due to the fact that the adult will reproduce every two or three days at a temperature of 25
degrees Celsius (Horning and Weber, 1985). During the 7-day test
period, therefore, potentially three broods are released per adult. Typically, six to ten neonates are released per brood
(Horning and Weber, 1985).
To summarize, the control sample data show that 11 adult Ceriodaphnia survived (a mortality rate of 8.33X), and that a
total of 176 neonates were borne to the 12 adult Ceriodaphnia
yielding an average of 14.67 neonates per adult. Likewise, the test sample (97.6X effluent) data corresponds to a mortality
rate of 83.33* (only 2 adult Ceriodaphnia surviving after 7- days), and an average of 11.75 neonates per adult. At this point, the analyzing laboratory performing the test must check that mortality of the control sample is less than or equal to 20X and that reproduction in the control sample exceeds 15 neonates
per initial female Ceriodaphnia. Should either one of these
conditions be violated, the test will be considered as abnormal and the test must be repeated. In the above example, because
only 14.67 neonates were borne per adult, the test violated the
control minimum reproduction criterion and, consequently, the test must be repeated.
Once the mortality and reproduction data are collected and tabulated and the control sample mortality and reproduction
criteria are met, then statistical comparisons for evaluating the control sample data with the test sample data can be utilized to determine if there is a significant difference in either
reproduction or mortality. Due to the tedious nature of the
calculations involved in computing the individual test statistics and the volume of toxicity test data to be analyzed, a
statistical spreadsheet was developed to perform the necessary calculations. Therefore, the remaining six sections of this chapter (sections 4.3 through 4.8) describe in detail, using examples, these statistical tests and the statistical
spreadsheet.
After November 1, 1989, Bartlett's Test for testing homogeneity of variance and Dunnett's Test for determining
significant difference in reproduction will no longer be utilized to statistically analyze chronic reproduction data (North
Carolina Division of Environmental Management, 1989). Instead, NCDEM will require chronic reproduction data to be analyzed by a T-Test as recommended in the USEPA's modified guidance docuement Short-Term Methods for Estimating the Chronic Toxicity sf.
Effluents and Receiving Waters to Freshwater Organisms. Second Edition, March 1989. Because the gathering of chronic toxicity data for this study ended as of August 1989, all chronic
reproduction data in this report has been analyzed by the statisitical methods in accordance with Figure 4-1 and as
explains the future T-Test statistical procedure, and presents a revised statistical spreadsheet incorporating the new procedural
changes.