Experimental systems

Salt exposure experimental systems were set up prior to field collection of organisms.

Artificial stream systems or channels (figure 2.7) were used for organisms collected from lotic environments (Balfour, Kat and Buffalo Rivers) (DWAF, 2000a). This protocol was adapted for the freshwater limpet Burnupia stenochorias (Ancylidae). Limpets were placed on plastic Petri-dishes and then submerged in the channel to allow the limpets an immediate and easy foothold on a substrate. Athericidae are known to be predacious and were therefore used in the lentic experimental system (50ml plastic vials, see below) to keep larvae separate from each other¹.

Using the underlying principles of toxicity testing described in DWAF (2000), an experimental system was designed to accommodate organisms collected from a lentic aquatic system, i.e. Dräger Dam. The system was designed for predacious damselflies (Coenagrionidae) which had to be accommodated individually to avoid cannabalism (Samway and Wilmot, 2003). The bottoms of 50ml plastic vials were sawed off, and a large hole was drilled in the lid (figure 2.8). The containers were then turned upside down and mesh gauze was secured in the bottom with the lid. Two further holes were drilled on opposite sides to allow several containers to be suspended on a single dowel stick, which was then suspended across the rim of a glass tank (figure 2.9) allowing three quarters of the container to be submerged in solution. Each tank was vigorously aerated to maintain oxygen levels in each vial over the experimental period. Glass tanks were replaced with plastic tubs (figure 2.10) to increase capacity of each experimental tank (from 24 to +40 vials) to allow for the variability introduced by two coenagrionid species. Vials were also used to conduct experiments using the Cloeon virgilae (Baetidae) collected from Dräger Dam with approximately five baetids allocated to one pill container during a toxicity test.

Similarly, glass tanks were used with larger 500ml plastic jars to provide leptoceridae and pleiidae with an area for swimming. Jars had two squares cut from both sides and a plastic mesh secured in their place, using silicon. The plastic mesh allowed for circulation. Two holes were drilled in the top of each jar. String was threaded through

1 At the time of the experiment being conducted, no lotic experimental system had been developed to keep predacious organisms separate from each other. The static system appeared adequate as no control mortality was observed and tolerance data met the assumptions of the quality criteria used in the study.

these holes and, using the string, the jars were secured to a single dowel stick with three jars per dowel stick.

Figure 2.7: Artificial stream systems or channels used for organisms collected from lotic enviornments

Figure 2.8: Two single 50ml plastic vials illustrating how gauze is secured into container

Figure 2.9: Lentic experimental system – aerated glass tanks with individual 50ml plastic vials (suspended on wooden dowel sticks) to

accommodate predacious organisms

Figure 2.10: Lentic experimental system – aerated plastic tubs containing 40+ 50ml plastic vials suspended by string on wooden dowel sticks

2.2.4 96 hour acute toxicity tests

De-chlorinated tap water was used as the diluent medium (prepared by passing tap water through a carbon filter) (DWAF, 2000a). Experimental vessels were filled with de-chlorinated tap water and allowed to cool to laboratory temperature to ensure constant temperature of diluent medium. Each lentic experimental vessel was aerated using air stones connected to air pumps with plastic tubing. After transportation, organisms were transferred into experimental vessels excluding damaged organisms and those with wingbuds (indicating last instar before emergence) and allowed 36 hour acclimation time (DWAF, 2000a). Prior to toxicants being added, experimental vessels were examined and dead organisms removed, recorded as acclimation mortalities and preserved in 70% ethanol to confirm identification. Vessels exhibiting >10% acclimation mortality were rejected from the experiment (DWAF, 2000a). After acclimation, toxicant solutions (NaCl or Na₂SO₄) were made up at selected concentrations using de-chlorinated tap water as the experimental medium. Where estimated LC50s for particular species were unknown, range finding tests were undertaken by selecting a wide range of concentrations to estimate the concentrations to be used for a definitive test. Details of number of organisms used per experimental vessel, number of experimental vessels used, concentrations used per experimental vessel and number

of controls used are provided for NaCl and Na₂SO₄ in tables 2.2a and 2.2b respectively.

Daily measurements of the following were taken in each experimental vessel, after organisms were exposed: electric conductivity (EC) and TDS using Amel 160 and Cyberscan 200 conductivity meters; pH using Cyberscan 10 and Beckman 10 pH meters; DO using WTW OXI92 dissolved oxygen meter and temperature using a thermometer (0-50°C). Immobility was used as a surrogate for death as the test endpoint (Cooney, 1995). Immobility was recorded at 12 hour intervals for a period of 96 hours. Immobile organisms were collected at each interval, labelled and preserved in 70% ethanol. Surviving organisms at the end of the 96 hour period were collected, labelled and preserved in 70% ethanol. Two water samples were collected from each experimental vessel at the end of the 96-hour period, one for macro-elements, nutrients, phosphate, ammonium and DO (each sample was preserved with mercury chloride) and the second for metal analysis. These were sent to the Department of Water Affairs and Forestry (Resource Quality Services-DWAF) in Pretoria for analysis.

Laboratory equipment was washed and cleaned in detergent and 2% hydrochloric acid.

Table 2.2a: Summary of number of organisms used per experimental vessel, number of controls used, number of experimental vessels and concentrations used per experimental vessel, for experiments using NaCl

Cloeon virgilae 50 3 8

500; 1500; 3000; 5000; 6000; 7000; 8000;

10 000

Plea pullula 30 1 6 500; 1500; 5000; 6000; 8000; 10 000

4

Coenagrioid sp. 40 1 9

5000; 10 000; 15 000; 20 000; 23 000; 27 000; 30 000; 35 000; 45 000

5 Leptoceris sp. 45

Plea pullula 30

6 Leptocerid sp. 45 1 9

500; 1000; 2000; 4000; 6000; 8000; 10 000; 15 000; 30 000

7

Plea pullula 45 1 10

100; 1000; 2000; 4000; 6000; 8000; 12 000; 15 000; 20 000; 40 000

100; 500; 1000; 2000; 3000; 5000; 6000;

8000; 10 000

Table 2.2b: Summary of number of organisms used per experimental vessel, number of controls used, number of experimental vessels and concentrations used per experimental vessel, for experiments using Na2SO4

3 Cloeon virgilae 40 3 8 8000

Plea pullula 30 1 6 100; 500; 1000; 2000; 3000; 4000

Coenagrionidae 12 1 5 5000; 8000; 15 000; 25 000; 40 000

4

Coenagrinoid sp. 40 1 9

5000; 15 000; 20 000; 24 000; 28 000; 32 000; 35 000; 40 000; 45 000

5 Leptoceris sp. 45

Plea pullula 30

6

Leptoceris sp. 45 1 9

500; 1000; 2000; 4000; 6000; 8000; 10 000; 15 000; 30 000

7

Plea pullula 45 1 10

100; 1000; 2000; 5000; 6000; 7000; 8000;

10 000; 15 000; 40 000

100; 1000; 3000; 4000; 5000; 6000; 7000;

8000; 12000